Frizzled-binding agents and uses thereof

ABSTRACT

Novel anti-cancer agents, including, but not limited to, antibodies and other polypeptides, that bind to human frizzled receptors are provided. Novel epitopes within the human frizzled receptors which are suitable as targets for anti-cancer agents are also identified. Methods of using the agents or antibodies, such as methods of using the agents or antibodies to inhibit Wnt signaling and/or inhibit tumor growth are further provided. Screening methods are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/078,728, filed Apr. 1, 2011, now U.S. Pat. No. 8,551,789, whichclaims the priority benefit of U.S. Provisional Application No.61/320,175, filed Apr. 1, 2010, each of which is incorporated byreference herein in its entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:sequencelisting_ascii.txt. Size: 182 kilobytes; and Date of Creation:Aug. 21, 2013) is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The field of this invention generally relates to polypeptides and otheragents that bind to human frizzled receptor(s), as well as to methods ofusing the polypeptides or other agents for the treatment of diseases,such as cancer.

BACKGROUND OF THE INVENTION

Cancer is one of the leading causes of death in the developed world,with over one million people diagnosed with cancer and 500,000 deathsper year in the United States alone. Overall it is estimated that morethan 1 in 3 people will develop some form of cancer during theirlifetime. There are more than 200 different types of cancer, four ofwhich-breast, lung, colorectal, and prostate—account for over half ofall new cases (Jemal et al., 2003, Cancer J. Clin. 53:5-26).

The Wnt signaling pathway has been identified as a potential target forcancer therapy. The Wnt signaling pathway is one of several criticalregulators of embryonic pattern formation, post-embryonic tissuemaintenance, and stem cell biology. More specifically. Wnt signalingplays an important role in the generation of cell polarity and cell fatespecification including self-renewal by stem cell populations.Unregulated activation of the Wnt pathway is associated with numeroushuman cancers where it can alter the developmental fate of tumor cellsto maintain them in an undifferentiated and proliferative state. Thuscarcinogenesis can proceed by usurping homeostatic mechanismscontrolling normal development and tissue repair by stem cells (reviewedin Reya & Clevers, 2005, Nature 434:843; Beachy et al., 2004, Nature432:324).

The Wnt signaling pathway was first elucidated in the Drosophiladevelopmental mutant wingless (wg) and from the murine proto-oncogeneint-1, now Wnt1 (Nusse & Varmus, 1982, Cell 31:99-109; Van Ooyen &Nusse, 1984, Cell 39:233-40; Cabrera et al., 1987. Cell 50:659-63;Rijsewijk et al., 1987, Cell 50:649-57). Wnt genes encode secretedlipid-modified glycoproteins of which 19 have been identified inmammals. These secreted ligands activate a receptor complex consistingof a Frizzled (Fzd) receptor family member and low-density lipoprotein(LDL) receptor-related protein 5 or 6 (LPR5/6). The Fzd receptors areseven transmembrane domain proteins of the G-protein coupled receptor(GPCR) superfamily and contain a large extracellular N-terminal ligandbinding domain with 10 conserved cysteines, known as a cysteine-richdomain (CRD) or Fri domain. There are ten human FZD receptors: FZD1-10.Different Fzd CRDs have different binding affinities for specific Wnts(Wu & Nusse, 2002. J. Biol. Chem. 277:41762-9), and Fzd receptors havebeen grouped into those that activate the canonical β-catenin pathwayand those that activate non-canonical pathways described below (Milleret al., 1999, Oncogene 18:7860-72). To form the receptor complex thatbinds the FZD ligands. FZD receptors interact with LRP5/6, single passtransmembrane proteins with four extracellular EGF-like domainsseparated by six YWTD amino acid repeats (Johnson et al., 2004, J. BoneMineral Res. 19:1749).

The canonical Wnt signaling pathway activated upon receptor binding ismediated by the cytoplasmic protein Dishevelled (Dsh) interactingdirectly with the Fzd receptor and results in the cytoplasmicstabilization and accumulation of β-catenin. In the absence of a Wntsignal, β-catenin is localized to a cytoplasmic destruction complex thatincludes the tumor suppressor proteins adenomatous polyposis coli (APC)and Axin. These proteins function as critical scaffolds to allowglycogen synthase kinase (GSK)-3β to bind and phosphorylate β-catenin,marking it for degradation via the ubiquitin/proteasome pathway.Activation of Dsh results in phosphorylation of GSK3β and thedissociation of the destruction complex. Accumulated cytoplasmicβ-catenin is then transported into the nucleus where it interacts withthe DNA-binding proteins of the Tcf/Lef family to activatetranscription.

In addition to the canonical signaling pathway, Wnt ligands alsoactivate 1-catenin-independent pathways (Veeman et al., 2003. Dev. Cell5:367-77). Non-canonical Wnt signaling has been implicated in numerousprocesses but most convincingly in gastrulation movements via amechanism similar to the Drosophila planar cell polarity (PCP) pathway.Other potential mechanisms of non-canonical Wnt signaling includecalcium flux, JNK, and both small and heterotrimeric G-proteins.Antagonism is often observed between the canonical and non-canonicalpathways, and some evidence indicates that non-canonical signaling cansuppress cancer formation (Olson & Gibo, 1998, Exp. Cell Res. 241:134;Topol et al., 2003, J. Cell Biol. 162:899-908). Thus, in certaincontexts, Fzd receptors act as negative regulators of the canonical Wntsignaling pathway. For example, FZD6 represses Wnt-3a-induced canonicalsignaling when co-expressed with FZD1 via the TAK1-NLK pathway (Golan etal., 2004, JBC 279:14879-88). Similarly, Fzd2 antagonized canonical Wntsignaling in the presence of Wnt-5a via the TAK1-NLK MAPK cascade(Ishitani et al. 2003. Mol. Cell. Biol. 23:131-9).

The canonical Wnt signaling pathway also plays a central role in themaintenance of stem cell populations in the small intestine and colon,and the inappropriate activation of this pathway plays a prominent rolein colorectal cancers (Reya & Clevers, 2005, Nature 434:843). Theabsorptive epithelium of the intestines is arranged into villi andcrypts. Stem cells reside in the crypts and slowly divide to producerapidly proliferating cells that give rise to all the differentiatedcell populations that move up out of the crypts to occupy the intestinalvilli. The Wnt signaling cascade plays a dominant role in controllingcell fates along the crypt-villi axis and is essential for themaintenance of the stem cell population. Disruption of Wnt signalingeither by genetic loss of Tcf7/2 by homologous recombination (Korinek etal., 1998, Nat. Genet. 19:379) or overexpression of Dickkopf-1 (Dkk1), apotent secreted Wnt antagonist (Pinto et al., 2003, Genes Dev.17:1709-13; Kuhnert et al. 2004, Proc. Nat'l. Acad. Sci. 101:266-71),results in depletion of intestinal stem cell populations.

Colorectal cancer is most commonly initiated by activating mutations inthe Wnt signaling cascade. Approximately 5-10% of all colorectal cancersare hereditary with one of the main forms being familial adenomatouspolyposis (FAP), an autosomal dominant disease in which about 80% ofaffected individuals contain a germline mutation in the adenomatouspolyposis coli (APC) gene. Mutations have also been identified in otherWnt pathway components including Axin and β-catenin. Individual adenomasare clonal outgrowths of epithelial cell containing a second inactivatedallele, and the large number of FAP adenomas inevitably results in thedevelopment of adenocarcinomas through addition mutations in oncogenesand/or tumor suppressor genes. Furthermore, activation of the Wntsignaling pathway, including gain-of-function mutations in APC andβ-catenin, can induce hyperplastic development and tumor growth in mousemodels (Oshima et al. 1997, Cancer Res. 57:1644-9; Harada et al., 1999,EMBO J. 18:5931-42).

A role for Wnt signaling in cancer was first uncovered with theidentification of Wnt1 (originally int1) as an oncogene in mammarytumors transformed by the nearby insertion of a murine virus (Nusse &Varmus, 1982, Cell 31:99-109). Additional evidence for the role of Wntsignaling in breast cancer has since accumulated. For instance,transgenic overexpression of β-catenin in the mammary glands results inhyperplasias and adenocarcinomas (Imbert et al., 2001, J. Cell Biol.153:555-68: Michaelson & Leder, 2001, Oncogene 20:5093-9) whereas lossof Wnt signaling disrupts normal mammary gland development (Tepera etal., 2003, J. Cell Sci. 116:1137-49; Hatsell et al., 2003, J. MammaryGland Biol. Neoplasia 8:145-58). More recently mammary stem cells havebeen shown to be activated by Wnt signaling (Liu et al., 2004, Proc.Nat'l Acad. Sci. 101:4158). In human breast cancer, β-cateninaccumulation implicates activated Wnt signaling in over 50% ofcarcinomas, and though specific mutations have not been identified,upregulation of Frizzled receptor expression has been observed (Brennan& Brown, 2004, J. Mammary Gland Neoplasia 9:119-31; Malovanovic et al.,2004, Int. J. Oncol. 25:1337-42).

FZD10, FZD8, FZD7, FZD4, and FZD5 are five often identified human Wntreceptors. Fzd10 is co-expressed with Wnt7b in the lungs, and celltransfection studies have demonstrated that the Fzd10/LRP5 co-receptoractivates the canonical Wnt signaling pathway in response to Wnt7b (Wanget al., 2005, Mol. Cell. Biol. 25:5022-30). FZD10 mRNA is upregulated innumerous cancer cell lines, including cervical, gastric, andglioblastoma cell lines, and in primary cancers including approximately40% of primary gastric cancers, colon cancers, and synovial sarcomas(Saitoh et al., 2002, Int. J. Oncol. 20:117-20; Terasaki et al., 2002,Int. J. Mol. Med. 9:107-12; Nagayama et al., 2005, Oncogene 1-12). FZD8is upregulated in several human cancer cell lines, primary gastriccancers, and renal carcinomas (Saitoh et al. 2001. Int. J. Oncol.18:991-96; Kirikoshi et al., 2001, Int. J. Oncol. 19:111-5: Janssens etal., 2004, Tumor Biol. 25:161-71). FZD7 is expressed throughout thegastrointestinal tract and is up-regulated in one out of six cases ofhuman primary gastric cancer (Kirikoshi et al., 2001, Int. J. Oncol.19:111-5). Expression of the FZD7 ectodomain by a colon cancer cell lineinduced morphological changes and decreased tumor growth in a xenograftmodel (Vincan et al. 2005, Differentiation 73:142-53). FZD5 plays anessential role in yolk sac and placental angiogenesis (Ishikawa et al.,2001, Dev. 128:25-33) and is upregulated in renal carcinomas inassociation with activation of Wnt/β-catenin signaling (Janssens et al.,2004, Tumor Biology 25:161-71). FZD4 is highly expressed in intestinalcrypt epithelial cells and is one of several factors that displaydifferential expression in normal versus neoplastic tissue (Gregorieffet al., 2005, Gastroenterology 129:626-38). The identification of FZDreceptors as markers of cancer stem cells thus makes these proteinsideal targets for cancer therapeutics.

SUMMARY OF THE INVENTION

The present invention provides novel agents that bind to one or morehuman frizzled receptors (FZDs), including, but not limited to,antibodies or other agents that bind two or more human frizzledreceptors, and methods of using the agents. The present inventionfurther provides novel polypeptides, such as antibodies that bind one ormore human frizzled receptors, fragments of such antibodies, and otherpolypeptides related to such antibodies. In certain embodiments, theagent, antibodies, other polypeptides, or agents that bind a FZD, bindto a region of the FZD referred to herein as the Biological Binding Site(BBS) that the inventors have now for the first time identified as atarget for inhibiting Wnt signaling and/or tumor growth. Antibodies andother polypeptides that comprise an antigen-binding site that binds morethan one FZD are also provided. Polynucleotides comprising nucleic acidsequences encoding the polypeptides are also provided, as are vectorscomprising the polynucleotides. Cells comprising the polypeptides and/orpolynucleotides of the invention are further provided. Compositions(e.g., pharmaceutical compositions) comprising the novel FZD-bindingagents or antibodies are also provided. In addition, methods of makingand using the novel FZD-binding agents or antibodies are also provided,such as methods of using the novel FZD-binding agents or antibodies toinhibit tumor growth and/or treat cancer.

Thus, in one aspect, the invention provides an agent that specificallybinds a human frizzled receptor. In certain embodiments, the agentinhibits the binding of a ligand (e.g., a Wnt) to the Biological BindingSite (BBS) of the human frizzled receptor. In certain embodiments, theagent binds to at least part of the Biological Binding Site (BBS) withinthe human frizzled receptor. In certain embodiments, the binding of theagent to the BBS results in an inhibition of Wnt signaling and/or tumorgrowth. In certain embodiments, the human frizzled receptor is FZD8 andthe agent binds to at least a part of (a) a conformational epitope ofFZD8 formed by amino acids 72(F), 74-75(PL), 78(I), 92(Y), 121-122(LM),and 129-132(WPDR (SEQ ID NO:70)); (b) a region of FZD8 consisting of thesequence QDEAGLEVHQFWPL (SEQ ID NO:67); and/or (c) a region of FZD8consisting of the sequence QYGFA (SEQ ID NO:66).

In certain embodiments, the human frizzled receptor bound by the agentis selected from the group consisting of FZD1, FZD2, FZD5, FZD7, orFZD8, and the agent binds to at least part of the sequenceQ(DE/ED)AGLEVHQF(Y/W)PL (SEQ ID NO:24) within the human frizzledreceptor. For instance, in certain embodiments, the human frizzledreceptor is FZD8 and the agent binds to at least part of the sequenceQDEAGLEVHQFWPL (SEQ ID NO:67) within FZD8. In certain embodiments, theagent binds to at least part of the sequence GLEVHQ (SEQ ID NO:25). Incertain embodiments, the human frizzled receptor is FZD1, FZD2, FZD3,FZD4, FZD5, FZD6. FZD7, FZD9, or FZD10, and the agent binds to at leastpart of a region of the human frizzled receptor corresponding to theregion of FZD8 consisting of QDEAGLEVHQFWPL (SEQ ID NO:67). In certainembodiments, the agent binds to at least part of a sequence(K/Q)(F/Y)GF(Q/A) (SEQ ID NO:69) within FZD1. FZD2, FZD5, FZD7, and/orFZD8. For example, in certain embodiments, the human frizzled receptoris FZD8 and the agent binds to at least part of a sequence QYGFA (SEQ IDNO:66) within FZD8. In certain alternative embodiments, the humanfrizzled receptor is FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD9, orFZD10, and the agent binds to at least part of a region of the humanfrizzled receptor corresponding to the region of FZD8 consisting ofQYGFA (SEQ ID NO:66). In certain embodiments, the agent specificallybinds to two or more, three or more, or four or more human frizzledreceptors. In certain embodiments, the agent specifically binds to humanfrizzled receptors comprising FZD5 and FZD8.

In certain alternative embodiments, the FZD-binding agent is an agentthat binds a human FZD receptor (e.g., FZD8) in the cleft of its BBS. Byway of nonlimiting example, the agent may bind one or more cleftresidues listed in Table 16. In certain embodiments, the agent binds L75of FZD8. In certain embodiments, the agent comprises a polypeptidecomprising a sequence having at least about 80% sequence identity to asequence selected from the group consisting of SEQ ID NOs:98-111 and SEQID NOs:113-116. In certain embodiments, the agent comprises SEQ IDNO:112. In some instances, the agent may comprise SEQ ID NO: 108.

In another aspect, the invention provides an agent that competes forspecific binding to a human frizzled receptor with an antibody (e.g., inan in vitro competitive binding assay), wherein the antibody comprises aheavy chain variable region comprising SEQ ID NO:10 and a light chainvariable region comprising SEQ ID NO:12 or SEQ ID NO:14. In certainembodiments, the antibody comprises a heavy chain variable regioncomprising SEQ ID NO:10 and a light chain variable region comprising SEQID NO:14. In certain embodiments, the agent competes for specificbinding to two or more, three or more, or four or more human frizzledreceptors. In certain embodiments, the agent competes for specificbinding to FZD1, FZD2, FZD5, FZD7, or FZD8.

In another aspect, the invention provides an agent that competes forspecific binding to a human FZD5 and/or FZD8 with an antibody thatcomprises a heavy chain variable region comprising SEQ ID NO:85 and alight chain variable region comprising SEQ ID NO:86.

In a further aspect, the invention provides a FZD-binding agent that (a)competes for binding to the BBS of FZD8 with a polypeptide and/or (b) iscapable of displacing from FZD8a polypeptide that is bound to the BBS ofFZD8. In certain embodiments, the polypeptide comprises a sequencehaving at least 80% identity with a sequence selected from the groupconsisting of SEQ ID NOs:98-111 and SEQ ID NOs:113-116. In certainalternative embodiments, the polypeptide comprises the sequence of SEQID NO: 112.

In another aspect, the invention provides an agent that specificallybinds to two or more human frizzled receptors. In certain embodiments,the two or more frizzled receptors comprise: (a) FZD1 and a secondfrizzled receptor selected from the group consisting of FZD2, FZD5.FZD7, and FZD8: (b) FZD2 and a second frizzled receptor selected fromthe group consisting of FZD5, FZD7, and FZD8; (c) FZD5 and FZD7; or (d)FZD7 and FZD8. In certain embodiments, the agent specifically bindsthree or more (i.e., 3, 4, or 5) human frizzled receptors, wherein thethree or more human frizzled receptors comprise FZD1, FZD2, FZD5, FZD7,and/or FZD8. In certain embodiments, the three or more human receptorscomprise FZD5 and FZD8. In certain embodiments, the three or more humanfrizzled receptors further comprise FZD3, FZD4, FZD6, FZD9, and/or FZD10.

In a further aspect, the invention provides a polypeptide thatspecifically binds a human frizzled receptor, wherein the polypeptidecomprises a heavy chain variable region comprising: (a) a heavy chainCDR1 comprising GFTFSHYTLS (SEQ ID NO:1), or a variant thereofcomprising 1, 2, 3, or 4 amino acid substitutions; (b) a heavy chainCDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO:2), or a variant thereofcomprising 1, 2, 3, or 4 amino acid substitutions; and/or (c) a heavychain CDR3 comprising NFIKYVFAN (SEQ ID NO:3), or a variant thereofcomprising 1, 2, 3, or 4 amino acid substitutions. In certainembodiments, the polypeptide specifically binds FZD1, FZD2, FZD5, FZD7,and/or FZD8. In certain embodiments, the polypeptide specifically bindstwo or more human frizzled receptors including FZD5 and FZD8. In certainembodiments, the amino acid substitutions are conservativesubstitutions.

In an additional aspect, the invention provides a polypeptide thatspecifically binds a human frizzled receptor, wherein the polypeptidecomprises a light chain variable region comprising: (a) a light chainCDR1 comprising SGDKLGKKYAS (SEQ ID NO:4) or SGDNIGSFYVH (SEQ ID NO:7),or a variant of SEQ ID NO:4 or SEQ ID NO:7 comprising 1, 2, 3, or 4amino acid substitutions; (b) a light chain CDR2 comprising EKDNRPSG(SEQ ID NO:5) or DKSNRPSG (SEQ ID NO:8), or a variant of SEQ ID NO:5 orSEQ ID NO:8 comprising 1, 2, 3, or 4 amino acid substitutions; and/or(c) a light chain CDR3 comprising SSFAGNSLE (SEQ ID NO:6) or QSYANTLSL(SEQ ID NO:9), or a variant of SEQ ID NO:6 or SEQ ID NO:9 comprising 1,2, 3, or 4 amino acid substitutions. In certain embodiments, thepolypeptide specifically binds FZD1, FZD2, FZD5, FZD7, and/or FZD8. Incertain embodiments, the polypeptide specifically binds two or morehuman frizzled receptors including FZD5 and FZD8. In certainembodiments, the amino acid substitutions are conservativesubstitutions.

In another aspect, the invention provides a polypeptide comprising: (a)a heavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO:1), a heavy chainCDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO:2), and a heavy chain CDR3comprising NFIKYVFAN (SEQ ID NO:3); and/or (b) a light chain CDR1comprising SGDKLGKKYAS (SEQ ID NO:4) or SGDNIGSFYVH (SEQ ID NO:7), alight chain CDR2 comprising EKDNRPSG (SEQ ID NO:5) or DKSNRPSG (SEQ IDNO:8), and a light chain CDR3 comprising SSFAGNSLE (SEQ ID NO:6) orQSYANTLSL (SEQ ID NO:9). In certain embodiments, the polypeptidespecifically binds a human frizzled receptor. In certain embodiments,the polypeptide specifically binds two or more (e.g., at least FZD5 andFZD8), three or more, or four or more human frizzled receptors.

In a further aspect, the invention provides an antibody thatspecifically binds a human frizzled receptor selected from the groupconsisting of FZD1, FZD2, FZD5, FZD7, and FZD8, wherein the antibodycomprises a heavy chain variable region comprising: (a) a heavy chainCDR1 comprising GFTFSHYTLS (SEQ ID NO:1), or a variant thereofcomprising 1, 2, 3, or 4 amino acid substitutions; a heavy chain CDR2comprising VISGDGSYTYYADSVKG (SEQ ID NO:2), or a variant thereofcomprising 1, 2, 3, or 4 amino acid substitutions; and a heavy chainCDR3 comprising NFIKYVFAN (SEQ ID NO:3), or a variant thereof comprising1, 2, 3, or 4 amino acid substitutions; and/or (b) a light chain CDR1comprising SGDKLGKKYAS (SEQ ID NO:4), SGDNIGSFYVH (SEQ ID NO:7), or avariant of either SEQ ID NO:4 or SEQ ID NO:7 comprising 1, 2, 3, or 4conservative amino acid substitutions; a light chain CDR2 comprisingEKDNRPSG (SEQ ID NO:5), DKSNRPSG (SEQ ID NO:8), or a variant of eitherSEQ ID NO:5 or SEQ ID NO:8 comprising 1, 2, 3, or 4 conservative aminoacid substitutions; and a light chain CDR3 comprising SSFAGNSLE (SEQ IDNO:6), QSYANTLSL (SEQ ID NO:9), or a variant of either SEQ ID NO:6 orSEQ ID NO:9 comprising 1, 2, 3, or 4 conservative amino acidsubstitutions. In certain embodiments, the antibody comprises (a) aheavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO:1), a heavy chain CDR2comprising VISGDGSYTYYADSVKG (SEQ ID NO:2), and a heavy chain CDR3comprising NFIKYVFAN (SEQ ID NO:3); and/or (b) a light chain CDR1comprising SGDKLGKKYAS (SEQ ID NO:4) or SGDNIGSFYVH (SEQ ID NO:7), alight chain CDR2 comprising EKDNRPSG (SEQ ID NO:5) or DKSNRPSG (SEQ IDNO:8), and a light chain CDR3 comprising SSFAGNSLE (SEQ ID NO:6) orQSYANTLSL (SEQ ID NO:9). In certain embodiments, the antibody comprises(a) a heavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO:1), a heavychain CDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO:2), and a heavy chainCDR3 comprising NFIKYVFAN (SEQ ID NO:3); and/or (b) a light chain CDR1comprising SGDNIGSFYVH (SEQ ID NO:7), a light chain CDR2 comprisingDKSNRPSG (SEQ ID NO:8), and a light chain CDR3 comprising QSYANTLSL (SEQID NO:9).

In another aspect, the invention provides a polypeptide comprising (a) apolypeptide having at least about 80% sequence identity to SEQ ID NO:10;and/or (b) a polypeptide having at least about 80% sequence identity toSEQ ID NO:12 or SEQ ID NO: 14. In certain embodiments, the inventionprovides a polypeptide comprising (a) a polypeptide having at leastabout 80% sequence identity to SEQ ID NO:10; and/or (b) a polypeptidehaving at least about 80% sequence identity to SEQ ID NO:14. In certainembodiments, the polypeptide specifically binds a human frizzledreceptor. In certain embodiments, the polypeptide specifically binds twoor more, three or more, or four or more human frizzled receptors. Incertain embodiments, the human frizzled receptor(s) are selected fromthe group consisting of FZD1, FZD2, FZD5, FZD7, and FZD8.

In still another aspect, the invention provides an agent such as anantibody that specifically binds human FZD5 and/or FZD8, wherein theantibody comprises: (a) a heavy chain CDR1 comprising GFTFSSYYIT (SEQ IDNO:77), or a variant thereof comprising 1, 2, 3, or 4 conservative aminoacid substitutions; a heavy chain CDR2 comprising TISYSSSNTYYADSVKG (SEQID NO:78), or a variant thereof comprising 1, 2, 3, or 4 conservativeamino acid substitutions; and a heavy chain CDR3 comprising SIVFDY (SEQID NO:79), or a variant thereof comprising 1, 2, 3, or 4 conservativeamino acid substitutions; and/or (b) a light chain CDR1 comprisingSGDALGNRYVY (SEQ ID NO:80), or a variant thereof comprising 1, 2, 3, or4 conservative amino acid substitutions; a light chain CDR2 comprisingSG (SEQ ID NO:81), or a variant thereof comprising 1, 2, 3, or 4conservative amino acid substitutions; and a light chain CDR3 comprisingGSWDTRPYPKY (SEQ ID NO:82), or a variant thereof comprising 1, 2, 3, or4 conservative amino acid substitutions. In certain embodiments, theantibody comprises: (a) a heavy chain CDR1 comprising GFTFSSYYIT (SEQ IDNO:77), a heavy chain CDR2 comprising TISYSSSNTYYADSVKG (SEQ ID NO:78),and a heavy chain CDR3 comprising SIVFDY (SEQ ID NO:79); and/or (b) alight chain CDR1 comprising SGDALGNRYVY (SEQ ID NO:80), a light chainCDR2 comprising SG (SEQ ID NO:81), and a light chain CDR3 comprisingGSWDTRPYPKY (SEQ ID NO:82).

In an additional aspect, the invention provides a polypeptide thatspecifically binds FZD5 and/or FZD8, wherein said polypeptide comprises:(a) a polypeptide having at least about 80% identity to SEQ ID NO:85;and/or (b) a polypeptide having at least about 80% identity to SEQ IDNO: 86.

In a further aspect, the invention provides an agent that competes forspecific binding to human FZD1, FZD2, FZD5, FZD7, and/or FZD8 with anyone of the following IgG antibodies: 18R8, 18R5, 18R4605, and 18R4805.

In a still further aspect, the invention provides an agent that competesfor specific binding to human FZD5 and/or FZD8 with the anti-FZD IgGantibody 44R24.

In certain embodiments of each of the aforementioned aspects, as well asother aspects described herein, the agent or polypeptide is an antibody.In certain alternative embodiments, the agent is not an antibody.

In certain embodiments of each of the aforementioned aspects, as well asother aspects described herein, the agent or polypeptide or antibodyspecifically binds to the extracellular domain (ECD) of the humanfrizzled receptor or receptors to which it binds. In certain embodimentsof each of the aforementioned aspects, as well as other aspectsdescribed herein, the agent or polypeptide or antibody specificallybinds to the Fri domain (Fri) of the human frizzled receptor orreceptors to which it binds.

In certain embodiments of each of the aforementioned aspects, as well asother aspects described elsewhere herein, an individual antigen-bindingsite of the antibody or other polypeptide specifically binds (or iscapable of binding) more than one human frizzled receptor.

In certain embodiments of each of the aforementioned aspects, as well asother aspects described herein, the agent or polypeptide or antibodyinhibits binding of a ligand to the human frizzled receptor(s). Incertain embodiments, the ligand is a Wnt, including, but not limited toWnt3a

In certain embodiments, of each of the aforementioned aspects, as wellas other aspects described herein, the agent or polypeptide or antibodythat binds to the FZD(s) is an antagonist of the FZD(s).

In certain embodiments of each of the aforementioned aspects, as well asother aspects described herein, the agent or polypeptide or antibodyinhibits Wnt signaling. In certain embodiments, the Wnt signaling thatis inhibited is canonical Wnt signaling. In some embodiments, the Wntsignaling that is inhibited by the FZD-binding agent is non-canonicalWnt signaling. In certain embodiments, the Wnt signaling isnon-canonical Wnt signaling.

In certain embodiments of each of the aforementioned aspects, as well asother aspects described herein, the FZD-binding agent or polypeptide orantibody inhibits tumor growth.

The invention further provides the antibodies 18R8, 18R5, 18R4605,44R24, and 18R4805, as well as fragments thereof.

The invention further provides compositions, such as pharmaceuticalcompositions, comprising a FZD-binding agent, polypeptide, or antibody.

Methods of inhibiting Wnt signaling (e.g., canonical Wnt signaling)and/or inhibiting tumor growth in a subject comprising administering atherapeutically effective amount of the FZD-binding agent or polypeptideor antibody are provided.

Methods of reducing the tumorigenicity of a tumor that comprises cancerstem cells are also provided. In certain embodiments, the methodscomprise administering a therapeutically effective amount of theFZD-binding agent or polypeptide or antibody to a subject comprising thetumor. In certain embodiments, the frequency of cancer stem cells in thetumor is reduced by administration of the antibody. In certainembodiments, administration of the FZD-binding agent results in thedifferentiation of tumorigenic cells in the tumor to a non-tumorigenicstate.

Also provided are methods of inducing cells in a tumor in a subject todifferentiate, said methods comprising administering a therapeuticallyeffective amount of the FZD-binding, agent, polypeptide, or antibody tothe subject.

Methods of treating cancer in a subject, comprising administering atherapeutically effective amount of the FZD-binding agent, polypeptide,or antibody to the subject are further provided.

In addition, methods of reducing myofibroblast activation in the stromaof a solid tumor, comprising contacting the stroma with an effectiveamount of the FZD-binding agent, polypeptide, or antibody are alsoprovided.

In other aspects, methods of treating a disease associated withWnt-signaling activation are provided. In certain embodiments, themethods comprise administering to a subject in need thereof an effectiveamount of a FZD-binding agent, polypeptide, or antibody. In certainembodiments, the disease is cancer. In certain alternative embodiments,the disease is polycystic kidney disease. In certain embodiments, theFZD-binding agent is a polypeptide that binds to the BBS of one or morehuman FZD receptors. In certain embodiments, the FZD-binding polypeptidecomprises a sequence having at least 80% identity with a sequenceselected from the group consisting of SEQ ID NOs:98-111 and SEQ IDNOs:113-116. In some embodiments, the FZD-binding polypeptide comprisesthe sequence of SEQ ID NO: 112. In certain alternative embodiments, theFZD-binding polypeptide is 18R5.

In certain embodiments, the methods comprising administration of theFZD-binding agent, polypeptide, or antibody further compriseadministering a second anti-cancer agent (e.g., a chemotherapeuticagent) to the subject. In certain embodiments, the second agent isgemcitabine, irinotecan, or paclitaxel. In certain embodiments, thesecond agent is an angiogenesis inhibitor and/or an inhibitor of Notchsignaling.

In another aspect, the invention provides a polypeptide comprising asequence selected from the group consisting of SEQ ID NOs: 10-15.Polypeptides comprising a sequence selected from the group consisting ofSEQ ID NOs: 85-86 are likewise provided. Polynucleotides comprisingnucleic acid sequences encoding such polypeptides are also provided.

In a further aspect, the invention provides a polynucleotide comprisinga sequence selected from the group consisting of SEQ ID NOs: 17-22.Polynucleotides comprising a sequence selected from the group consistingof SEQ ID NOs: 87-90, 92, and 94-95 are further provided.

In a still further aspect, the invention provides a polynucleotide thatcomprises a polynucleotide that hybridizes to a polynucleotide selectedfrom the group consisting of SEQ ID NOs: 17, 19, 21, 87-90, 92. and94-95, or a polynucleotide encoding a polypeptide selected from thegroup consisting of SEQ ID NOs: 10, 12, 14, and 85-86 under conditionsof high stringency. In certain embodiments, the invention comprises apolynucleotide that hybridizes to a polynucleotide consisting of asequence SEQ ID NOs: 17, 19, or 21, or a polynucleotide encoding SEQ IDNOs: 10, 12, or 14 under conditions of high stringency.

In certain embodiments of each of the aforementioned aspects, as well asother aspects described herein, the agent or polypeptide or antibody orpolynucleotide is isolated. In certain embodiments, the agent orpolypeptide or antibody or polynucleotide is substantially pure.

The present invention further provides a Wnt gene signature useful forthe identification of tumors and/or patients likely to respond totreatment with a FZD-binding agent (e.g., an antagonist of a humanfrizzled receptor and/or an inhibitor of Wnt signaling) or otherinhibitors of Wnt signaling. Methods of using the Wnt gene signature toselect patients for treatment with a FZD-binding agent or otherinhibitor of Wnt signaling are also provided. In certain embodiments,the methods involve the assessment of the level of one or more genes inthe Wnt gene signature. Methods of screening drug candidates againsttumors identified using the Wnt gene signature are also provided.Arrays, kits, and other compositions useful in the methods are alsoprovided.

The present invention also provides methods of screening for inhibitorsof Wnt signaling, potential drug candidates, or other agents. Thesemethods include, but are not limited to, methods comprising comparingthe levels of one or more differentiation markers in a first solid tumorthat has been exposed to the agent relative to the levels of the one ormore differentiation markers in a second solid tumor that has not beenexposed to the agent. In certain embodiments, these methods includecomprising (a) exposing a first solid tumor, but not a second solidtumor, to the agent; (b) assessing the levels of one or moredifferentiation markers in the first and second solid tumors; and (c)comparing the levels of the one or more differentiation markers in thefirst and second solid tumors. In certain alternative embodiments, themethods involve the use of FZD receptors or derivatives of FZD receptorswhich comprise one or more substitutions in the BBS or polypeptides orother agents which have already been identified as binding to portionsof the BBS. Agents identified using the screening methods, or having thecharacteristics of agents identified using the screening methods, arelikewise provided, as are methods of using such agents to inhibit Wntsignaling and/or in therapy.

In one aspect the invention provides a method of screening a testcompound for activity or possible activity as an inhibitor of Wntsignaling. In certain embodiments, the method comprises comparing (a)the binding affinity of the test compound for a first polypeptidecomprising the Fri domain of a human FZD receptor (e.g., FZD8) havingone or more substitutions in its BBS with (b) the binding affinity ofthe compound for a second polypeptide comprising the wild-type Fridomain. If the test compound has greater affinity for the secondpolypeptide than the first polypeptide, the compound is identified as aninhibitor of Wnt signaling or a possible inhibitor of Wnt signaling. Incertain embodiments, the method further comprises the additional stepsof first determining the binding affinity of the test compound for thefirst and second polypeptides. Reagents useful in such methods includingthe mutant Fri domain are also provided. Agents identified as inhibitorsof Wnt signaling (or at least potential Wnt inhibitors) by such methodsare likewise provided.

In another aspect, the invention provides a method of screening a testcompound for activity or possible activity as an inhibitor of Wntsignaling that comprises determining whether the test compound cancompete with a FZD-binding agent (that binds the BBS) for binding to ahuman FZD receptor. In certain embodiments, the ability of the compoundto compete for binding with the polypeptide indicates the compound hasactivity or possible activity as an inhibitor of Wnt signaling.

In another aspect, the invention provides alternative methods ofscreening a test compound for activity or possible activity as aninhibitor of Wnt signaling. In certain embodiments the method comprisesdetermining whether the test compound can displace a FZD-binding agent(that binds the BBS) from the human FZD receptor to which it is bound.If the test compound can displace the FZD-binding agent, the compoundhas activity or possible activity as an inhibitor of Wnt signaling.Reagents useful in the above-indicated competitive binding anddisplacement assays, including, without limitation, polypeptidescomprising a sequence that is at least about 80% identical to a sequenceselected from the group consisting of SEQ ID NOs:98-111 and SEQ IDNOs:113-116, are provided. Agents identified as inhibitors of Wntsignaling (or at least potential Wnt inhibitors) by such competitivebinding and displacement assays are likewise provided.

Where aspects or embodiments of the invention are described in terms ofa Markush group or other grouping of alternatives, the present inventionencompasses not only the entire group listed as a whole, but each memberof the group individually and all possible subgroups of the main group,but also the main group absent one or more of the group members. Thepresent invention also envisages the explicit exclusion of one or moreof any of the group members in the claimed invention.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1. 18R8 binds multiple human frizzled receptors. FACS analysisdemonstrated that 18R8 binds to FZD1, FZD2, FZD5, FZD7, and FZD8sequences on transiently transfected HEK293 cells. FACS plots are shownfor the binding of 18R8 to HEK293 cells transfected with expressionvectors encoding the indicated FZD and an expression vector for GFP.18R8 binding is indicated by elevated staining within the co-transfected(GFP positive) cell population. FACS plots for FZD1, FZD2, FZD5, FZD7and FZD8 are boxed with thick line to highlight the FZDs showing bindingto 18R8.

FIG. 2. Anti-FZD antibody 18R5 binds multiple human frizzled receptorson cells. FACS analysis demonstrated that 18R5, like 18R8, binds toFZD1, FZD2, FZD5. FZD7, and FZD8 sequences on cells. Antibodies 18R8 and18R5 were incubated at a range of concentrations with the HEK293 cellsoverexpressing the indicated FZD and antibody binding was assessed byflow cytometry. While both 18R8 and 18R5 bind to FZD1. FZD2, FZD5, FZD7and FZD8, 18R5 binds with greater affinity.

FIG. 3. Luciferase reporter assays were carried out in STF293 cells thatstably express 8xTCF promoter element linked to luciferase. Cells weretreated with conditioned medium containing Wnt3A as well as a range ofconcentrations of 18R8 and 18R5 and then assayed 18 hours later usingthe Dual-Glo luciferase assay reporter system (Promega). The resultsdemonstrate that both 18R8 and 18R5 inhibit Wnt signaling and that 18R5binds with greater affinity than 18R8.

FIG. 4. 18R8 blocks TCF signaling by multiple Wnts. Luciferase reporterassays were carried out in STF293 cells that stably express 8xTCFpromoter element linked to luciferase. Various Wnt-overexpressing cellswere generated by transfecting HEK293 cells (ATCC) with expressionvectors encoding indicated Wnt proteins using Fugene 6 (Roche). STF293cells were treated with 18R8 and added Wnts overexpressed HEK293 cellsand assayed 18 hours later using the Dual-Glo luciferase assay reportersystem.

FIG. 5. 18R8 directly inhibits Wnt binding to FZD. Luciferase reporterassays were carried out in STF293 cells that stably express 8xTCFpromoter element. A mixture containing Wnt3A conditioned medium,purified FZD8-Fc, and/or 18R8 were co-incubated as indicated for 2 hrsat 4° C. with/without protein A sepharose beads. After the incubation,the protein A sepharose beads were removed and added to STF293 cells.The treated STF293 cells were assayed 18 hours later using the Dual-Gloluciferase assay reporter system. This experiment shows that in theabsence of 18R8, Fzd8-Fc is able to inhibit the ability of Wnt3A tostimulate signaling, but that 18R8 is able to block the ability of FZD8to bind Wnt3A, as evidenced by the restoration of signaling when thesepharose A beads are used to remove the FZD8-Fc (and 18R8) from theco-incubation.

FIG. 6. FACS analysis of binding of 18R8 to mutant FZD8 compared towild-type FZD8. To assess the epitope of 18R8 on FZD, epitope mappingstudies were performed. An expression construct that enabled theexpression of the Fri domain of human FZD8 with an N-terminal FLAG tag,and a C-terminal CD4 transmembrane domain and intracellular domain wasused in transient transfection studies together with an expressionvector encoding GFP. Variants of this expression vector were alsoprepared which contained selected amino acid substitutions within theFZD8 sequence to encode the amino acids at the corresponding positionwithin the Fri domain of other particular FZDs not bound by 18R8. Theability of 18R8 to bind to these variant FZD8 sequences was thenassessed by flow cytometry. Certain positions including amino acids66-71 and 126-127 of FZD8 were found to be required for 18R8 binding asindicated by reduced staining within the co-transfected (GFP positive)cell population. The region of the FACS plot showing binding of 18R8 tothe cotransfected cell population is highlighted by a box and the box isshown in thick lines for those amino acid substitutions showing markedlyreduced 18R8 binding.

FIG. 7. 18R5 and 18R8 are shown to have a similar binding epitope onhuman FZD8. The ability of 18R5 to bind to a similar epitope as 18R8 wasassessed by flow cytometry using a series of amino acid variantsprevious shown to disrupt binding of 18R8. Positions including aminoacids 66-71 and 126-127 of FZD8 were found to be required for bindingboth 18R8 and 18R5 as indicated by reduced staining within theco-transfected (GFP positive) cell population. The region of the FACSplot showing binding of 18R8 to the cotransfected cell population ishighlighted by a box and the box is shown in thick lines for those aminoacid substitutions showing markedly reduced 18R8 and 18R5 binding.

FIG. 8. Comparison of the amino acid sequences of portions of the Fridomain sequences of the human frizzled receptors. Sites with conservedresidues are shaded in black; sites with similar amino acid residues areshaded in gray. The FZD epitope of 18R8 and 18R5 contains the regionsdenoted by underline and labeled as “top edge” and “bottom edge.” Theterms “top edge” and “bottom edge” reflect the recognition, based onexamination of Fri domain crystal structure, that these regions flank acleft on the surface of the FZD protein. This cleft contains multiplehighly conserved residues. The amino acids that comprise this cleft arehighlighted by carrot symbols above each corresponding position withinthe alignment. This region has not previously been ascribed specificfunction. The discovery of antibodies that bind to this region and thediscovery that these antibodies inhibit Wnt binding and Wnt signaling aswell as our recognition of the conserved nature of this cleft haveenabled us to identify this region as a key functional aspect of the FZDproteins.

FIG. 9. The Biological Binding Site (BBS) of FZD. Shown are images ofthe structure of a Fzd fri domain. The images are based on analysis ofthe previously reported crystal structure of mouse FZD8 (Dann C E etal., Nature 412 (6842) 86-90, 2001) and analysis done using the softwareprogram Pymol. Shown in the upper left image is a surface view of theFZD Fri domain with the region of the Fzd protein comprising thebiological binding site (BBS) that the inventors have discovered(encircled by a white oval). This is the region bound by the antibodies18R8 and 18R5. This region contains structural elements we term the “topedge” the “bottom edge” and the “cleft.” Each of these is highlighted indarker surface coloration in separate images at the bottom of the panel.The upper right image highlights in darker surface the residues that areconserved in nine or ten of the ten human Fzd family members andhighlights the recognition that a distinct grouping of these residuesoccurs within the center of the “cleft” region flanked by the epitopethat binds antibodies that inhibit Fzd function.

FIG. 10. Prevention of Wnt-dependent tumor growth by anti-FZD mAb.NOD/SCID mice injected with 50,000 MMTV WNT1 tumor derived cells andtumor growth was monitored weekly until growth was detected, then tumorgrowth was measured twice a week. Ten mice with established tumors weretreated with either 18R8 or a control antibody as a control. Tumorgrowth in animals treated with 18R8 was virtually eliminated compared tothat observed in animals treated with control antibody.

FIG. 11. Reduction of OMP-C28 xenograft tumor growth by combinationtreatment of 18R5 and irinotecan. NOD/SCID mice were injected with10,000 OMP-C28 colon tumor cells, and on day 24, mice with tumors ofaverage volume of 129 mm³ were randomized and the indicated treatmentswere initiated. Tumor growth was monitored weekly. Tumor growth inanimals treated with 18R5 was significantly reduced. Further, thecombination of 18R5 and irinotecan was significantly reduced overtreatment with either agent alone.

FIG. 12. Reduction of OMP-PN4 xenograft tumor growth by combinationtreatment of 18R5 and gemcitabine. NOD/SCID mice injected with 50,000OMP-PN4 pancreatic tumor cells and on day 38, mice with tumors ofaverage volume of about 120 mm³ were randomized, and the indicatedtreatments were initiated two days later. Tumor growth in animalstreated with the combination of 18R5 and gemcitabine was significantlyreduced over treatment with gemcitabine alone.

FIG. 13. Heavy chain and light chain amino acid sequences for 18R8 and18RS, including VH and VL sequences.

FIG. 14. Nucleotide sequences encoding the heavy chain and VH sequencesof 18R8 and 18R5.

FIG. 15. Nucleotide sequences encoding the light chain and VL sequencesof 18R8 and 18R5.

FIG. 16. Amino acid sequence of FZD7 ECD Fc protein and nucleotidesequence encoding same.

FIG. 17. Amino acid sequences of human FZD1 (SEQ ID NO:26), theextracellular domain (ECD) of FZD1 (SEQ ID NO:27, shown as underlinedamino acids 1-321 of SEQ ID NO:26), and the Fri domain of FZD1 (SEQ IDNO:28).

FIG. 18. Amino acid sequences of human FZD2 (SEQ ID NO:30), theextracellular domain (ECD) of FZD2 (SEQ ID NO:31, shown as underlinedamino acids 1-250 of SEQ ID NO:30), and the Fri domain of FZD2 (SEQ IDNO:32).

FIG. 19. Nucleotide sequence encoding human FZD1.

FIG. 20. Nucleotide sequence encoding human FZD2.

FIG. 21. Amino acid sequences of human FZD3 (SEQ ID NO:34), theextracellular domain (ECD) of FZD3 (SEQ ID NO:35, shown as underlinedamino acids 1-204 of SEQ ID NO:34), and the Fri domain of FZD3 (SEQ IDNO:36).

FIG. 22. Amino acid sequences of human FZD4 (SEQ ID NO:38), theextracellular domain (ECD) of FZD4 (SEQ ID NO:39, shown as underlinedamino acids 1-224 of SEQ ID NO:38), and the Fri domain of FZD4 (SEQ IDNO:40).

FIG. 23. Nucleotide sequence encoding human FZD3.

FIG. 24. Nucleotide sequence encoding human FZD4.

FIG. 25. Amino acid sequences of human FZD5 (SEQ ID NO:42), theextracellular domain (ECD) of FZD5 (SEQ ID NO:43, shown as underlinedamino acids 1-233 of SEQ ID NO:42), and the Fri domain of FZD5 (SEQ IDNO:44).

FIG. 26. Amino acid sequences of human FZD6 (SEQ ID NO:46), theextracellular domain (ECD) of FZD6 (SEQ ID NO:47, shown as underlinedamino acids 1-207 of SEQ ID NO:46), and the Fri domain of FZD6 (SEQ IDNO:48).

FIG. 27. Nucleotide sequence encoding human FZD5.

FIG. 28. Nucleotide sequence encoding human FZD6.

FIG. 29. Amino acid sequences of human FZD7 (SEQ ID NO:50), theextracellular domain (ECD) of FZD7 (SEQ ID NO:51, shown as underlinedamino acids 1-255 of SEQ ID NO:50), and the Fri domain of FZD7 (SEQ IDNO:52).

FIG. 30. Amino acid sequences of human FZD8 (SEQ ID NO:54), theextracellular domain (ECD) of FZD8 (SEQ ID NO:55, shown as underlinedamino acids 1-277 of SEQ ID NO:54), and the Fri domain of FZD8 (SEQ IDNO:56).

FIG. 31. Nucleotide sequence encoding human FZD7.

FIG. 32. Nucleotide sequence encoding human FZD8.

FIG. 33. Amino acid sequences of human FZD9 (SEQ ID NO:58), theextracellular domain (ECD) of FZD9 (SEQ ID NO:59, shown as underlinedamino acids 1-230 of SEQ ID NO:58), and the Fri domain of FZD9 (SEQ IDNO:60).

FIG. 34. Amino acid sequences of human FZD10 (SEQ ID NO:62), theextracellular domain (ECD) of FZD10 (SEQ ID NO:63, shown as underlinedamino acids 1-227 of SEQ ID NO:62), and the Fri domain of FZD10 (SEQ IDNO:64).

FIG. 35. Nucleotide sequence encoding human FZD9.

FIG. 36. Nucleotide sequence encoding human FZD 10.

FIG. 37. Reduction of PE-13 breast tumor growth with combinationtreatment of 18R5 antibody and paclitaxel. NOD/SCID mice were injectedwith 10,000 PE-13 breast tumor cells and on day 22, mice with tumors ofaverage volume of about 120 mm³ were randomized. The mice were thentreated with either a control antibody (“Control Ab”), 18R5 antibody(“Anti-FZD”), paclitaxel (“Taxol”), or a combination of 18R5 antibodyplus paclitaxel (“Anti-FZD+Taxol”). Treatment with 18R5 antibody incombination with paclitaxel resulted in anti-tumor activity.

FIG. 38. Breast tumor growth in individual animals treated with thecombination of 18R5 antibody plus paclitaxel. Treatment with 18R5antibody in combination with paclitaxel resulted in regression ofestablished breast tumors.

FIG. 39. Flow cytometry analysis of colorectal tumor cells followingtreatment with control antibody, 18R5 antibody, irinotecan, or both 18R5antibody and irinotecan.

FIG. 40. Tumor growth in mice following implantation of 30, 90, 270, or810 tumor cells obtained from mice that had been treated for 41 dayswith either control antibody (“Control”), 18R5 antibody (“Anti-FZD”),gemcitabine (“Gemcitabine”), or the combination of 18R5 plus gemcitabine(“Combination”).

FIG. 41. Cancer stem cell (CSC) frequency in PN-4 pancreatic tumorsfollowing treatment with control antibody (“Control Ab”), 18R5 antibodyalone (“Anti-FZD”), gemcitabine alone (“Gemcitabine”), or thecombination of 18R5 antibody and gemcitabine (“Combination”), asdetermined by limiting dilution analysis.

FIG. 42. Chromatogram gene expression in pancreatic tumor cells thathave been treated with control antibody (“LZ-1”), 18R5 antibody alone(“18R5”), gemcitabine alone (“Gem”), or a combination of gemcitabine and18R5 (“Combo”).

FIG. 43. Treatment with anti-FZD antibody 18R5 promotes differentiationof tumor cells into non-proliferative mucinous cells. NOD/SCID mice wereinjected with 50,000 OMP-PN13 pancreatic tumor cells and on day 23, micewith tumors of average volume of about 107 mm³ were randomized, andtreatment with control antibody or 18R5 was initiated four days later.After 20 days tumors were collected and sectioned. Tumor sections from18R5 treated mice or control antibody treated mice were stained withalcian blue stain to reveal mucinous cells and by immunohistochemistrywith antibody to ki67 to reveal proliferative cells. Exemplary mucinouscells and proliferative cells are highlighted by arrows.

FIG. 44. Anti-FZD antibody 18R5, both alone and in combination withTaxol® (paclitaxel), inhibits OMP-LU24 xenograft tumor growth. Micebearing OMP-LU24 human lung tumors were treated with either controlantibody (“Control Ab”), anti-FZD 18R5 (“18R5”), Taxol® (“Taxol”) or thecombination of 18R5 plus Taxol, (“18R5+Taxol”).

FIG. 45. Anti-FZD antibody 18R5, both alone and in combination withAvastin® (bevacizumab), inhibits OMP-LU33 xenograft tumor growth. Micebearing OMP-LU33 human lung tumors were treated with either controlantibody (squares). Avastin® (triangles pointing up), anti-FZD 18R5(triangles pointing down), or the combination of 18R5 plus Avastin®(circles).

FIG. 46. The combination of anti-FZD antibody 18R5 with Herceptin®(trastuzumab) inhibits the growth of T3 xenograft tumor growth. Micecarrying T3 human breast tumors were treated with either controlantibody (squares), anti-FZD 18R5 (triangles), Herceptin® (filledcircles), or the combination of 18R5 plus Herceptin® (open circles).

FIG. 4. 7, 18R5 and 44R24Fzd binding profiles. Dose-reponse curverepresenting the binding of each mAb to Fzd 1, 2, 5, 7 and 8.

FIG. 48. Inhibition of Wnt3a-induced reporter activity in STF cells by18R5 and 44R24 (dose response curves).

FIG. 49. Inhibition of basal level of axin2 gene expression by 18R5 and44R24.

FIG. 50. THC detection of Muc16 in 18R5 and 44R24-treated OMP-PN13tumors.

FIGS. 51A and B. Inhibition of smooth muscle actin in 18R5-treatedpancreatic tumor. FIG. 51A. ACTA2 gene expression levels as detected bymicroarray. FIG. 51B. SMA detection on control mAb (upper panel) and18R5 (lower panel)-treated OMP-PN4 tumors.

FIGS. 52A-C. Testing the tumorgenicity of 18R5-induced Muc16+OMP-PNI3cells. FIG. 52A. FACS plot of lin-depleted OMP-PN13 tumor cells stainedfor Muc16. The 2 sorted populations are circled. FIG. 52B.Representative pictures of tumors resulting from the injection ofMuc16-(upper panel) and Muc16+(lower panel) cells. FIG. 52C. Tumorgrowth curves.

FIG. 53. Inhibition of tumor recurrence by anti-FZD mAb 18R5 in PE13breast tumor xenograft.

FIG. 54. Reduction of breast cancer stem cell frequency by anti-FZDmAb18R5.

FIG. 55. Inhibition of tumor recurrence by anti-FZD mAb 18R5 in PN4pancreatic tumor xenograft.

FIG. 56. Inhibition of tumor growth by anti-FZD mAb 44R24 in combinationwith gemcitabine in PN4 pancreatic tumor xenograft.

FIG. 57. FACS analysis of binding of anti-FZD mAb 44R24 to mutant FZD8relative to wild-type FZD8. The region of the FACS plot showing bindingof 44R24 to the cotransfected cell population is highlighted by a box.The box for those amino acid substitutions showing markedly reduced44R24 binding is marked with an arrow.

FIG. 58. Anti-Tumor activity of anti-FZD Antibodies 44R24 and 18R5 inC28 colon tumor xenografts.

FIG. 59. Induction of cytokeratin 7 expression in C28 colon tumorxenografts treated with anti-FZD antibodies 44R24 or 18R5.

FIGS. 60A-C. Residues within the OMP-18R5 binding site impact Wntbinding. FIG. 60A. Shown are images of the structure of a Fzd fridomain. The images are based on analysis of the previously reportedcrystal structure of mouse FZD8 (Dann C E et al., Nature 412 (6842)86-90, 2001) and analysis done using the software program Pymol. Thisregion contains structural elements we term the “top edge” the “bottomedge” and the “cleft”. Shown in the upper left image is a surface viewof the FZD fri domain with the region of the Fzd protein highlightingresidues conserved in nine or ten of the human frizzled receptors. Theupper right highlights some of the residues bound by 18R5. FIG. 60B.Shown are images depicting the position of four amino acid substitutionswithin the cleft region by grey highlight. The position and amino acidsubstitution (single letter amino acid code) within human FZD8 islisted. FIG. 60C. The ability of FZD8-Fc fusion proteins including thevariants containing amino acid substitutions within the cleft region toact as soluble decoy molecules to bind Wnt3A is examined. Luciferasereporter assays were carried out in STF293 cells that stably express8xTCF promoter element. Cells were exposed to Wnt3A-containing mediumand a range of dilutions of medium contain FZD8-Fc variants for 18 hoursand then luciferase activity was measured with the Dual-Glo luciferaseassay reporter system (Promega). The results demonstrate that FZD8-Fcvariants that contain amino acid substitutions with the cleft region areless able to bind to Wnt3A and inhibit its ability to elicit Wntsignaling with the STF293 cells.

FIG. 61. Identification of peptides that bind to FZD. Shown is ELISAdata testing the ability of individual phage clones expressing peptidesto bind to FZD8-Fc or a negative control protein, DLL4-Fc. Arrowshighlight several positive clones by way of example.

FIG. 62. Analysis of FZD binding peptide sequences. Shown are peptidesequences found to bind specifically to FZD. Both cyclic and linearpeptide sequences were identified. The cyclic peptides show a clearsequence similarity enabling the definition of a consensus sequence.Because the peptide library is unlikely to contain all possible aminoacid variations related to this consensus, it is likely that additionalvariants outside of this consensus would also be capable of binding toFZD. The linear peptides did not display strong consensus similarity.However, several of these sequences (e.g. clone#2 DTLSALIERGLM (SEQ IDNO: 113)) were potent FZD binders to the 18R5 binding region of FZD andbound within the cleft region found to be important for Wnt-FZDinteraction.

FIG. 63. FZD binding peptides bind within the “cleft” in 18R5binding/Wnt binding region. To examine the ability of the FZD bindingpeptides to interact with the “cleft” within the 18R5 binding region,ELISA was conducted testing the ability of individual phage clonesexpressing peptides to bind to FZD8-FC or a FZD8-Fc variant (Mut2)containing an amino acid substitution within the cleft, or a negativecontrol protein, DLL4-Fc. Many of the peptides bound the FZD8-Mut2protein much less well than wild-type FZD8. Several peptides, ashighlighted by arrows, displayed little if any binding to the FZD8-Mut2protein indicating that they bound specifically within the “cleft”. Anexample of such a peptide, clone#2, had the sequence DTLSALIERGLM (SEQID NO:113).

FIG. 64. Inhibition of Wnt signaling by peptides that bind to FZD. Shownare the results of Wnt dependent luciferase reporter studies examiningthe ability of two peptides (clones #2 and #24) to inhibit Wntsignaling. The two peptides were generated by chemical synthesis. Thepeptides were dissolved into dimethyl sulfoxide (DMSO) and then dilutedinto the reported assay at the concentrations indicated. The reporterassay utilized HEK293 cells transfected with a plasmid containing theWnt responsive 8xTCF luciferase reporter. Cells were exposed to Wnt3Aand the indicated peptide and then incubated for 16 hours. Wnt activitywas determined using the Dual-glo luciferase assay (Promega).

FIG. 65. Minimum Fri domains of human FZD1-5.

FIG. 66. Minimum Fri domains of human FZD6-10.

FIG. 67. Anti-FZD antibody 18R5 inhibits the ability of several Wnts toinduce canonical signaling.

FIG. 68. Binding of 18R5 to FZD inhibits the interaction of WNT withFZD.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel agents, including, but not limitedto polypeptides such as antibodies, that bind to one or more humanfrizzled receptors (FZDs). Related polypeptides and polynucleotides,compositions comprising the FZD-binding agents, and methods of makingthe FZD-binding agents are also provided. Methods of using the novelFZD-binding agents, such as methods of inhibiting tumor growth and/ortreating cancer, are further provided.

The invention is based, in part, on the identification of a regionwithin human frizzled receptors that is a suitable target forFZD-binding, anti-cancer agents. Two anti-FZD antibodies. 18R8 and 18R5,were found to specifically bind to FZD7, but also to cross-react withFZD1, FZD2, FZD5, and FZD8 (Examples 1 and 2, below). In vitroexperiments with the 18R8 antibody indicated that the antibody iscapable of inhibiting Wnt signaling (Example 3, below) and inhibitingbinding of Wnt ligands to FZD8 (Example 4, below). The 18R5 antibody hasalso been demonstrated to likewise be capable of inhibiting Wntsignaling in cell-based assays (Examples 3 and 20, below). In vivoexperiments with the 18R5 antibody demonstrated that the antibody iscapable of inhibiting tumor growth or recurrence (Examples 7, 17, and23, below). The inventors have also shown the anti-FZD antibody 18R5 tobe capable of reducing the frequency of cancer stem cells in tumors(Examples, 8 and 23, below) and inducing the differentiation and/orreducing the tumorigenicity of tumor cells (Examples 16, 21, 22, and 25,below). Epitope mapping experiments with these active 18R8 and 18R5antibodies indicated that both of the antibodies bind to at least partof the sequence GLEVHQ (SEQ ID NO:25) and at least part of the sequenceYGFA (SEQ ID NO:74) within FZD8 (Example 5, below). In light of thedemonstrated biological activity of these two antibodies, the crystalstructure of mouse Frizzled 8 (Dann et al., Nature, 412: 86-90 (2001))was analyzed and an extracellular region of frizzled proteins comprisingthese sequences that had not previously been ascribed any specificfunction was identified for the first time as playing an importantfunctional role in FZD biology and Wnt signaling (Example 6). Thisregion of human frizzled receptors, designated the Biological BindingSite (BBS), is a suitable target for anti-cancer therapies.

In addition, a third antibody, 44R24, was found to specifically bind tohuman FZD5 and FZD8 (Example 19, below). This antibody has also beenshown to be capable of inhibiting Wnt signaling in cell-based assays(Example 20, below) and of anti-tumor efficacy in vivo (Examples 23 and25, below). Like treatment with the anti-FZD antibodies 18R8 and 18R5,treatment of a tumor with 44R24 resulted in increased levels of adifferentiation marker in the tumor (Example 25, below). Epitope mappinghas also shown that the epitope of the anti-FZD antibody 44R24 overlapswith that of the anti-FZD antibodies 18R8 and 18R5. More specifically.44R24 has been shown to bind to at least part of the region YGFA (SEQ IDNO:74) in the BBS (Example 24, below).

I. DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below.

The term “antibody” means an immunoglobulin molecule that recognizes andspecifically binds to a target, such as a protein, polypeptide, peptide,carbohydrate, polynucleotide, lipid, or combinations of the foregoingthrough at least one antigen recognition site within the variable regionof the immunoglobulin molecule. As used herein, the term “antibody”encompasses intact polyclonal antibodies, intact monoclonal antibodies,antibody fragments (such as Fab, Fab′, F(ab′)2, and Fv fragments),single chain Fv (scFv) mutants, multispecific antibodies such asbispecific antibodies generated from at least two intact antibodies,chimeric antibodies, humanized antibodies, human antibodies, fusionproteins comprising an antigen determination portion of an antibody, andany other modified immunoglobulin molecule comprising an antigenrecognition site so long as the antibodies exhibit the desiredbiological activity. An antibody can be of any the five major classes ofimmunoglobulins: IgA. IgD, IgE, IgG, and IgM, or subclasses (isotypes)thereof (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on theidentity of their heavy-chain constant domains referred to as alpha,delta, epsilon, gamma, and mu, respectively. The different classes ofimmunoglobulins have different and well known subunit structures andthree-dimensional configurations. Antibodies can be naked or conjugatedto other molecules such as toxins, radioisotopes, etc.

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limited toFab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, single chainantibodies, and multispecific antibodies formed from antibody fragments.

A “monoclonal antibody” refers to a homogeneous antibody populationinvolved in the highly specific recognition and binding of a singleantigenic determinant, or epitope. This is in contrast to polyclonalantibodies that typically include different antibodies directed againstdifferent antigenic determinants. The term “monoclonal antibody”encompasses both intact and full-length monoclonal antibodies as well asantibody fragments (such as Fab, Fab′, F(ab′)2, Fv), single chain (scFv)mutants, fusion proteins comprising an antibody portion, and any othermodified immunoglobulin molecule comprising an antigen recognition site.Furthermore, “monoclonal antibody” refers to such antibodies made in anynumber of manners including but not limited to by hybridoma, phageselection, recombinant expression, and transgenic animals.

The term “humanized antibody” refers to forms of non-human (e.g. murine)antibodies that are specific immunoglobulin chains, chimericimmunoglobulins, or fragments thereof that contain minimal non-human(e.g., murine) sequences. Typically, humanized antibodies are humanimmunoglobulins in which residues from the complementary determiningregion (CDR) are replaced by residues from the CDR of a non-humanspecies (e.g. mouse, rat, rabbit, hamster) that have the desiredspecificity, affinity, and capability (Jones et al., 1986, Nature.321:522-525; Riechmann et al., 1988, Nature. 332:323-327; Verhoeyen etal. 1988, Science. 239:1534-1536). In some instances, the Fv frameworkregion (FR) residues of a human immunoglobulin are replaced with thecorresponding residues in an antibody from a non-human species that hasthe desired specificity, affinity, and capability. The humanizedantibody can be further modified by the substitution of additionalresidues either in the Fv framework region and/or within the replacednon-human residues to refine and optimize antibody specificity,affinity, and/or capability. In general, the humanized antibody willcomprise substantially all of at least one, and typically two or three,variable domains containing all or substantially all of the CDR regionsthat correspond to the non-human immunoglobulin whereas all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody can also comprise at least aportion of an immunoglobulin constant region or domain (Fc), typicallythat of a human immunoglobulin. Examples of methods used to generatehumanized antibodies are described in U.S. Pat. No. 5,225,539.

The term “human antibody” means an antibody produced by a human or anantibody having an amino acid sequence corresponding to an antibodyproduced by a human made using any technique known in the art. Thisdefinition of a human antibody includes intact or full-lengthantibodies, fragments thereof, and/or antibodies comprising at least onehuman heavy and/or light chain polypeptide such as, for example, anantibody comprising murine light chain and human heavy chainpolypeptides.

The term “chimeric antibodies” refers to antibodies wherein the aminoacid sequence of the immunoglobulin molecule is derived from two or morespecies. Typically, the variable region of both light and heavy chainscorresponds to the variable region of antibodies derived from onespecies of mammals (e.g. mouse, rat, rabbit, etc) with the desiredspecificity, affinity, and capability while the constant regions arehomologous to the sequences in antibodies derived from another (usuallyhuman) to avoid eliciting an immune response in that species.

The term “epitope” or “antigenic determinant” are used interchangeablyherein and refer to that portion of an antigen capable of beingrecognized and specifically bound by a particular antibody. When theantigen is a polypeptide, epitopes can be formed both from contiguousamino acids and noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids are typicallyretained upon protein denaturing, whereas epitopes formed by tertiaryfolding are typically lost upon protein denaturing. An epitope typicallyincludes at least 3, and more usually, at least 5 or 8-10 amino acids ina unique spatial conformation.

That an antibody “specifically binds” to an epitope or protein meansthat the antibody reacts or associates more frequently, more rapidly,with greater duration, with greater affinity, or with some combinationof the above to an epitope or protein than with alternative substances,including unrelated proteins. In certain embodiments, “specificallybinds” means, for instance, that an antibody binds to a protein with aK_(D) of about 0.1 mM or less, but more usually less than about 1 μM. Incertain embodiments, “specifically binds” means that an antibody bindsto a protein at times with a K_(D) of at least about 0.1 μM or less, andat other times at least about 0.01 μM or less. Because of the sequenceidentity between homologous proteins in different species, specificbinding can include an antibody that recognizes a particular proteinsuch as a frizzled receptor in more than one species. Likewise, becauseof homology between different FZD receptors (e.g., FZD5 and FZD8) incertain regions of the polypeptide sequences of the receptors, specificbinding can include an antibody (or other polypeptide or agent) thatrecognizes more than one frizzled receptor. It is understood that anantibody or binding moiety that specifically binds to a first target mayor may not specifically bind to a second target. As such, “specificbinding” does not necessarily require (although it can include)exclusive binding, i.e. binding to a single target. Thus, an antibodymay, in certain embodiments, specifically bind to more than one target(e.g., human FZD1, FZD2. FZD5, FZD7, and/or FZD8). In certainembodiments, the multiple targets may be bound by the sameantigen-binding site on the antibody. For example, an antibody may, incertain instances, comprise two identical antigen-binding sites, each ofwhich specifically binds two or more human frizzled receptors (e.g.,human FZD1, FZD2, FZD5, FZD7, and/or FZD8). In certain alternativeembodiments, an antibody may be bispecific and comprise at least twoantigen-binding sites with differing specificities. By way ofnon-limiting example, a bispecific antibody may comprise oneantigen-binding site that recognizes an epitope on one frizzledreceptor, such as human FZD5, and further comprises a second, differentantigen-binding site that recognizes a different epitope on a secondfrizzled receptor, such as human FZD8. Generally, but not necessarily,reference to binding means specific binding.

A polypeptide, antibody, polynucleotide, vector, cell, or compositionwhich is “isolated” is polypeptide, antibody, polynucleotide, vector,cell, or composition which is in a form not found in nature. Isolatedpolypeptides, antibodies, polynucleotides, vectors, cell or compositionsinclude those which have been purified to a degree that they are nolonger in a form in which they are found in nature. In some embodiments,an antibody, polynucleotide, vector, cell, or composition which isisolated is substantially pure.

As used herein, “substantially pure” refers to material which is atleast 50% pure (i.e., free from contaminants), more preferably at least90% pure, more preferably at least 95% pure, more preferably at least98% pure, more preferably at least 99% pure.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals in which a population of cells arecharacterized by unregulated cell growth. Examples of cancer include,but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia. More particular examples of such cancers include squamous cellcancer, small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma and various types of head and neckcancers.

“Tumor” and “neoplasm” refer to any mass of tissue that result fromexcessive cell growth or proliferation, either benign (noncancerous) ormalignant (cancerous) including pre-cancerous lesions.

The terms “cancer stem cell,” “tumor stem cell,” or “solid tumor stemcell” are used interchangeably herein and refer to a population of cellsfrom a solid tumor that: (1) have extensive proliferative capacity; 2)are capable of asymmetric cell division to generate one or more kinds ofdifferentiated progeny with reduced proliferative or developmentalpotential; and (3) are capable of symmetric cell divisions forself-renewal or self-maintenance. These properties of “cancer stemcells,” “tumor stem cells,” or “solid tumor stem cells” confer on thosecancer stem cells the ability to form palpable tumors upon serialtransplantation into an immunocompromised mouse compared to the majorityof tumor cells that fail to form tumors. Cancer stem cells undergoself-renewal versus differentiation in a chaotic manner to form tumorswith abnormal cell types that can change over time as mutations occur.

The terms “cancer cell,” “tumor cell,” and grammatical equivalents referto the total population of cells derived from a tumor or a pre-cancerouslesion, including both non-tumorigenic cells, which comprise the bulk ofthe tumor cell population, and tumorigenic stem cells (cancer stemcells). As used herein, the term “tumor cell” will be modified by theterm “non-tumorigenic” when referring solely to those tumor cellslacking the capacity to renew and differentiate to distinguish thosetumor cells from cancer stem cells.

The term “tumorigenic” refers to the functional features of a solidtumor stem cell including the properties of self-renewal (giving rise toadditional tumorigenic cancer stem cells) and proliferation to generateall other tumor cells (giving rise to differentiated and thusnon-tumorigenic tumor cells) that allow solid tumor stem cells to form atumor. These properties of self-renewal and proliferation to generateall other tumor cells confer on cancer stem cells the ability to formpalpable tumors upon serial transplantation into an immunocompromisedmouse compared to non-tumorigenic tumor cells, which are unable to formtumors upon serial transplantation. It has been observed thatnon-tumorigenic tumor cells may form a tumor upon primarytransplantation into an immunocompromised mouse after obtaining thetumor cells from a solid tumor, but those non-tumorigenic tumor cells donot give rise to a tumor upon serial transplantation.

The term “subject” refers to any animal (e.g. a mammal), including, butnot limited to humans, non-human primates, rodents, and the like, whichis to be the recipient of a particular treatment. Typically, the terms“subject” and “patient” are used interchangeably herein in reference toa human subject.

“Pharmaceutically acceptable salt” refers to a salt of a compound thatis pharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound.

“Pharmaceutically acceptable excipient, carrier or adjuvant” refers toan excipient, carrier or adjuvant that can be administered to a subject,together with at least one antibody of the present disclosure, and whichdoes not destroy the pharmacological activity thereof and is nontoxicwhen administered in doses sufficient to deliver a therapeutic amount ofthe compound.

“Pharmaceutically acceptable vehicle” refers to a diluent, adjuvant,excipient, or carrier with which at least one antibody of the presentdisclosure is administered.

The term “therapeutically effective amount” refers to an amount of anantibody, polypeptide, polynucleotide, small organic molecule, or otherdrug effective to “treat” a disease or disorder in a subject or mammal.In the case of cancer, the therapeutically effective amount of the drugcan reduce the number of cancer cells; reduce the tumor size; inhibit orstop cancer cell infiltration into peripheral organs including, forexample, the spread of cancer into soft tissue and bone; inhibit andstop tumor metastasis; inhibit and stop tumor growth; relieve to someextent one or more of the symptoms associated with the cancer; reducemorbidity and mortality; improve quality of life; decreasetumorigenicity, tumorgenic frequency, or tumorgenic capacity of a tumor;reduce the number or frequency of cancer stem cells in a tumor;differentiate tumorigenic cells to a non-tumorigenic state; or acombination of such effects. To the extent the drug prevents growthand/or kills existing cancer cells, it can be referred to as cytostaticand/or cytotoxic.

Terms such as “treating” or “treatment” or “to treat” or “alleviating”or “to alleviate” refer to both 1) therapeutic measures that cure, slowdown, lessen symptoms of, and/or halt progression of a diagnosedpathologic condition or disorder and 2) prophylactic or preventativemeasures that prevent and/or slow the development of a targetedpathologic condition or disorder. Thus, those in need of treatmentinclude those already with the disorder; those prone to have thedisorder; and those in whom the disorder is to be prevented. In certainembodiments, a subject is successfully “treated” for cancer according tothe methods of the present invention if the patient shows one or more ofthe following: a reduction in the number of or complete absence ofcancer cells; a reduction in the tumor size; inhibition of or an absenceof cancer cell infiltration into peripheral organs including, forexample, the spread of cancer into soft tissue and bone; inhibition ofor an absence of tumor metastasis; inhibition or an absence of tumorgrowth; relief of one or more symptoms associated with the specificcancer; reduced morbidity and mortality; improvement in quality of life;reduction in tumorigenicity, tumorgenic frequency, or tumorgeniccapacity, of a tumor; reduction in the number or frequency of cancerstem cells in a tumor; differentiation of tumorigenic cells to anon-tumorigenic state; or some combination of effects.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase. A polynucleotidemay comprise modified nucleotides, such as methylated nucleotides andtheir analogs. If present, modification to the nucleotide structure maybe imparted before or after assembly of the polymer. The sequence ofnucleotides may be interrupted by non-nucleotide components. Apolynucleotide may be further modified after polymerization, such as byconjugation with a labeling component. Other types of modificationsinclude, for example, “caps”, substitution of one or more of thenaturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.methyl phosphonates, phosphotriesters, phosphoamidates, cabamates, etc.)and with charged linkages (e.g., phosphorothioates, phosphorodithioates,etc.), those containing pendant moieties, such as, for example, proteins(e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine,etc.), those with intercalators (e.g., acridine, psoralen, etc.), thosecontaining chelators (e.g., metals, radioactive metals, boron, oxidativemetals, etc.), those containing alkylators, those with modified linkages(e.g., alpha anomeric nucleic acids, etc.), as well as unmodified formsof the polynucleotide(s). Further, any of the hydroxyl groups ordinarilypresent in the sugars may be replaced, for example, by phosphonategroups, phosphate groups, protected by standard protecting groups, oractivated to prepare additional linkages to additional nucleotides, ormay be conjugated to solid supports. The 5′ and 3′ terminal OH can bephosphorylated or substituted with amines or organic capping groupmoieties of from 1 to 20 carbon atoms. Other hydroxyls may also bederivatized to standard protecting groups. Polynucleotides can alsocontain analogous forms of ribose or deoxyribose sugars that aregenerally known in the art, including, for example, 2′-O-methyl-.2′-O-allyl. 2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs,.alpha.-anomeric sugars, epimeric sugars such as arabinose, xyloses orlyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclicanalogs and abasic nucleoside analogs such as methyl riboside. One ormore phosphodiester linkages may be replaced by alternative linkinggroups. These alternative linking groups include, but are not limitedto, embodiments wherein phosphate is replaced by P(O)S (“thioate”),P(S)S (“dithioate”), “(O)NR₂ (“amidate”), P(O)R′, P(O)OR′, CO or CH₂(“formacetal”), in which each R or R′ is independently H or substitutedor unsubstituted alkyl (1-20 C) optionally containing an ether (—O—)linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not alllinkages in a polynucleotide need be identical. The precedingdescription applies to all polynucleotides referred to herein, includingRNA and DNA.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. The variable regions of the heavy andlight chain each consist of four framework regions (FR) connected bythree complementarity determining regions (CDRs) also known ashypervariable regions. The CDRs in each chain are held together in closeproximity by the FRs and, with the CDRs from the other chain, contributeto the formation of the antigen-binding site of antibodies. There are atleast two techniques for determining CDRs: (1) an approach based oncross-species sequence variability (i.e., Kabat et al. Sequences ofProteins of Immunological Interest, (5th ed., 1991, National Institutesof Health, Bethesda Md.)); and (2) an approach based on crystallographicstudies of antigen-antibody complexes (Al-lazikani et al (1997) J.Molec. Biol. 273:927-948)). In addition, combinations of these twoapproaches are sometimes used in the art to determine CDRs.

The term “vector” means a construct, which is capable of delivering, andpreferably expressing, one or more gene(s) or sequence(s) of interest ina host cell. Examples of vectors include, but are not limited to, viralvectors, naked DNA or RNA expression vectors, plasmid, cosmid or phagevectors, DNA or RNA expression vectors associated with cationiccondensing agents. DNA or RNA expression vectors encapsulated inliposomes, and certain eukaryotic cells, such as producer cells.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art. Itis understood that, because the polypeptides of this invention are basedupon antibodies, in certain embodiments, the polypeptides can occur assingle chains or associated chains.

The terms “identical” or percent “identity” in the context of two ormore nucleic acids or polypeptides, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides or amino acid residues that are the same, when compared andaligned (introducing gaps, if necessary) for maximum correspondence, notconsidering any conservative amino acid substitutions as part of thesequence identity. The percent identity may be measured using sequencecomparison software or algorithms or by visual inspection. Variousalgorithms and software are known in the art that may be used to obtainalignments of amino acid or nucleotide sequences. One such non-limitingexample of a sequence alignment algorithm is the algorithm described inKarlin et al, 1990, Proc. Natl. Acad. Sci., 87:2264-2268, as modified inKarlin et al., 1993, Proc. Natl. Acad. Sci. 90:5873-5877, andincorporated into the NBLAST and XBLAST programs (Altschul et al., 1991,Nucleic Acids Res., 25:3389-3402). In certain embodiments, Gapped BLASTmay be used as described in Altschul et al., 1997, Nucleic Acids Res.25:3389-3402. BLAST-2, WU-BLAST-2 (Altschul et al., 1996, Methods inEnzymologyv, 266:460-480), ALIGN. ALIGN-2 (Genentech. South SanFrancisco, Calif.) or Megalign (DNASTAR) are additional publiclyavailable software programs that can be used to align sequences. Incertain embodiments, the percent identity between two nucleotidesequences is determined using the GAP program in GCG software (e.g.using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90and a length weight of 1, 2, 3, 4, 5, or 6). In certain alternativeembodiments, the GAP program in the GCG software package, whichincorporates the algorithm of Needleman and Wunsch (J. Mol. Biol.(48):444-453 (1970)) may be used to determine the percent identitybetween two amino acid sequences (e.g., using either a Blossum 62 matrixor a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and alength weight of 1, 2, 3, 4, 5). Alternatively, in certain embodiments,the percent identity between nucleotide or amino acid sequences isdetermined using the algorithm of Myers and Miller (CABIOS, 4:11-17(1989)). For example, the percent identity may be determined using theALIGN program (version 2.0) and using a PAM120 with residue table, a gaplength penalty of 12 and a gap penalty of 4. Appropriate parameters formaximal alignment by particular alignment software can be determined byone skilled in the art. In certain embodiments, the default parametersof the alignment software are used. In certain embodiments, thepercentage identity “X” of a first amino acid sequence to a secondsequence amino acid is calculated as 100×(Y/Z), where Y is the number ofamino acid residues scored as identical matches in the alignment of thefirst and second sequences (as aligned by visual inspection or aparticular sequence alignment program) and Z is the total number ofresidues in the second sequence. If the length of a first sequence islonger than the second sequence, the percent identity of the firstsequence to the second sequence will be longer than the percent identityof the second sequence to the first sequence.

As a non-limiting example, whether any particular polynucleotide has acertain percentage sequence identity (e.g., is at least 80% identical,at least 85% identical, at least 90% identical, and in some embodiments,at least 95%, 96%. 97%. 98%, or 99% identical) to a reference sequencecan, in certain embodiments, be determined using the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group. University Research Park, 575 Science Drive, Madison,Wis. 53711). Bestfit uses the local homology algorithm of Smith andWaterman, Advances in Applied Mathematics 2: 482 489 (1981), to find thebest segment of homology between two sequences. When using Bestfit orany other sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set such that thepercentage of identity is calculated over the full length of thereference nucleotide sequence and that gaps in homology of up to 5% ofthe total number of nucleotides in the reference sequence are allowed.

In some embodiments, two nucleic acids or polypeptides of the inventionare substantially identical, meaning they have at least 70%, at least75%, preferably at least 80%, more preferably at least 85%, morepreferably at least 90%, and in some embodiments at least 95%, 96%, 97%.98%, 99% nucleotide or amino acid residue identity, when compared andaligned for maximum correspondence, as measured using a sequencecomparison algorithm or by visual inspection. Preferably, identityexists over a region of the sequences that is at least about 10,preferably about 20, more preferable about 40-60 residues in length orany integral value therebetween, preferably over a longer region than60-80 residues, more preferably at least about 90-100 residues, and mostpreferably the sequences are substantially identical over the fulllength of the sequences being compared, such as the coding region of anucleotide sequence for example.

A “conservative amino acid substitution” is one in which one amino acidresidue is replaced with another amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). For example, substitution of aphenylalanine for a tyrosine is a conservative substitution. Preferably,conservative substitutions in the sequences of the polypeptides andantibodies of the invention do not abrogate the binding of thepolypeptide or antibody containing the amino acid sequence, to theantigen(s), i.e., the one or more human frizzled receptors to which thepolypeptide or antibody binds. Methods of identifying nucleotide andamino acid conservative substitutions which do not eliminate antigenbinding are well-known in the art (see, e.g., Brummell et al., Biochem.32: 1180-1 187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884(1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:.412-417 (1997)).

As used in the present disclosure and claims, the singular forms “a,”“an,” and “the” include plural forms unless the context clearly dictatesotherwise.

It is understood that wherever embodiments are described herein with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

The term “and/or” as used in a phrase such as “A and/or B” herein isintended to include both “A and B,” “A or B,” “A,” and “B.” Likewise,the term “and/or” as used in a phrase such as “A, B, and/or C” isintended to encompass each of the following embodiments: A, B, and C; A,B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B(alone); and C (alone).

“Conditions of high stringency.” may be identified by those that: (1)employ low ionic strength and high temperature for washing, for example0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecylsulfate at 50° C.; (2) employ during hybridization a denaturing agent,such as formamide, for example. 50% (v/v) formamide with 0.1% bovineserum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodiumphosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodiumcitrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl,0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodiumpyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42°C. in 0.2×SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C., followed by a high-stringency wash consisting of 0.1×SSC containingEDTA at 55° C.

II. FZD-BINDING AGENTS

The present invention provides agents that specifically bind one or morehuman frizzled receptors (FZDs). These agents are referred to herein as“FZD-binding agents.” In certain embodiments, the agents specificallybind two, three, four, five, six, seven, eight, nine, or ten frizzledreceptors. The human frizzled receptor or receptors bound by the agentmay be selected from the group consisting of FZD1, FZD2, FZD3, FZD4,FZD5. FZD6, FZD7. FZD8, FZD9, and FZD10. In certain embodiments, the oneor more human frizzled receptors comprise FZD1, FZD2, FZD5, FZD7, and/orFZD8. In certain embodiments, the one or more human frizzled receptorscomprise FZD7. In certain embodiments, the one or more human frizzledreceptors comprise FZD5 and/or FZD8. In certain embodiments, the agentspecifically binds FZD1, FZD2, FZD5, FZD7, and FZD8. The full-lengthamino acid (aa) and nucleotide (nt) sequences for FZD1-10 are known inthe art and also provided herein as SEQ ID NO:26 (FZD1 aa), SEQ ID NO:30(FZD2 aa). SEQ ID NO:34 (FZD3 aa), SEQ ID NO:38 (FZD4 aa), SEQ ID NO:42(FZD5 aa). SEQ ID NO:46 (FZD6 aa), SEQ ID NO:50 (FZD7 aa). SEQ ID NO:54(FZD8 aa), SEQ ID NO:58 (FZD9 aa), SEQ ID NO:62 (FZD10 aa), SEQ ID NO:29(FZD1 nt), SEQ ID NO:33 (FZD2 nt), SEQ ID NO:37 (FZD3 nt), SEQ ID NO:41(FZD4 nt), SEQ ID NO:45 (FZD5 nt), SEQ ID NO:49 (FZD6 nt). SEQ ID NO:53(FZD7 nt). SEQ ID NO:57 (FZD8 nt), SEQ ID NO:61 (FZD9 nt), and SEQ IDNO:65 (FZD10 nt).

In certain embodiments, the antibody or other polypeptide or agentdescribed herein specifically binds FZD7. In certain embodiments, thatantibody, polypeptide, or agent may further specifically bind orcross-react with one or more additional human frizzled receptors.

In certain embodiments, the antibody or other polypeptide or agentdescribed herein specifically binds FZD5. In certain embodiments, thatantibody, polypeptide, or agent may further specifically bind orcross-react with one or more additional human frizzled receptors.

In certain embodiments, the agent specifically binds to two or morehuman frizzled receptors. In certain embodiments, the two or more humanfrizzled receptors are selected from the group consisting of FZD2, FZD5,FZD7, and FZD8. In certain embodiments, the two or more frizzledreceptors comprise FZD1 and a second frizzled receptor selected from thegroup consisting of FZD2, FZD5. FZD7, and FZD8. In certain embodiments,the two or more frizzled receptors comprise FZD2 and a second frizzledreceptor selected from the group consisting of FZD1, FZD5, FZD7, andFZD8. In certain embodiments, the two or more frizzled receptorscomprise FZD5 and a second frizzled receptor selected from the groupconsisting of FZD1, FZD2, FZD7, and FZD8. In certain embodiments, thetwo or more frizzled receptors comprise both FZD5 and FZD8. In certainembodiments, the two or more frizzled receptors comprise FZD7 and asecond frizzled receptor selected from the group consisting of FZD1,FZD2, FZD5, and FZD8.

In certain embodiments, the agent specifically binds to three or morehuman frizzled receptors. In certain embodiments, the three or morehuman frizzled receptors comprise three or more frizzled receptorsselected from the group consisting of FZD1, FZD2. FZD5, FZD7, and FZD8.In certain embodiments, the agent further specifically binds to one ormore additional human frizzled receptors.

In certain embodiments, the agent or antibody specifically binds to theextracellular domain (ECD) within the one or more human frizzledreceptors to which it binds. Sequences of the extracellular domain ofeach of the human frizzled receptors are known in the art and are alsoprovided as SEQ ID NO:27 (FZD1 ECD), SEQ ID NO:31 (FZD2 ECD). SEQ IDNO:35 (FZD3 ECD), SEQ ID NO:39 (FZD4 ECD), SEQ ID NO:43 (FZD5 ECD), SEQID NO:47 (FZD6 ECD), SEQ ID NO:51 (FZD7 ECD), SEQ ID NO:55 (FZD8 ECD).SEQ ID NO:59 (FZD9 ECD), and SEQ ID NO:63 (FZD10 ECD).

In certain embodiments, the agent or antibody specifically binds to theFri domain (FRI) (also known as the cysteine-rich domain (CRD)) withinthe human frizzled receptor(s) to which it binds. Sequences of the Fridomain of each of the human frizzled receptors are known in the art andare also provided as SEQ ID NO:28 (FZD1 FRI), SEQ ID NO:32 (FZD2 FRI),SEQ ID NO:36 (FZD3 FRI). SEQ ID NO:40 (FZD4 FRI), SEQ ID NO:44 (FZD5FRI), SEQ ID NO:48 (FZD6 FRI), SEQ ID NO:52 (FZD7 FRI), SEQ ID NO:56(FZD8 FRI), SEQ ID NO:60 (FZD9 FRI), and SEQ ID NO:64 (FZD10 FRI).

In certain embodiments, an individual antigen-binding site of aFZD-binding antibody or polypeptide described herein is capable ofbinding (or binds) the one, two, three, four, or five (or more) humanfrizzled receptors. In certain embodiments, an individualantigen-binding site of the FZD-binding antibody or polypeptide iscapable of specifically binding one, two, three, four, or five humanfrizzled receptors selected from the group consisting of FZD1, FZD2,FZD5, FZD7, and FZD8. In certain embodiments, an individual binding siteof the antibody or polypeptide specifically binds to at least FZD5 andFZD8.

In certain embodiments, the FZD-binding agent or antibody binds to oneor more (for example, two or more, three or more, or four or more) humanfrizzled receptors with a dissociation constant (K_(D)) of about 1 μM orless, about 100 nM or less, about 40 nM or less, about 20 nM or less, orabout 10 nM or less. For example, in certain embodiments, a FZD-bindingagent or antibody described herein that binds to more than one FZD,binds to those FZDs with a K_(D) of about 100 nM or less, about 20 nM orless, or about 10 nM or less. In certain embodiments, the FZD-bindingagent or antibody binds to each of one or more (e.g., 1, 2, 3, 4, or 5)of the following FZDs with a dissociation constant of about 40 nM orless: FZD1, FZD2, FZD5, FZD7, and FZD8. In certain embodiments, theFZD-binding agent or antibody binds to each of one or more of thefollowing FZDs with a dissociation constant of about 10 nM or less:FZD1, FZD2, FZD5, FZD7, and FZD8. In certain embodiments, theFZD-binding agent or antibody binds to each of the following FZDs with adissociation constant of about 10 nM or less: FZD1, FZD2. FZD5, FZD7,and FZD8. In certain embodiments, the dissociation constant of the agentor antibody to a particular FZD is the dissociation constant determinedusing an FZD-Fc fusion protein comprising the FZD extracellular domainor Fri domain immobilized on a Biacore chip.

In certain embodiments, the FZD-binding agent or antibody binds to eachof one or more of the following FZDs with a dissociation constantbetween about 1 nM and about 10 nM: FZD1, FZD2, FZD5, FZD7, and FZD8.Alternatively, the FZD-binding agent or antibody binds to two or more(or three or more) of the following FZDs with a dissociation constantbetween about 1 nM and about 10 nM: FZD1, FZD2, FZD5, FZD7, and FZD8. Incertain further embodiments, the FZD-binding agent or antibody binds toeach of both FZD5 and FZD8 with a dissociation constant between about 1nM and about 10 nM.

In certain embodiments, the FZD-binding agent or antibody binds to FZD8with a dissociation contant of at least about 3 nM. In certainembodiments, the FZD-binding agent or antibody binds to FZD8 with adissociation constant between about 3 nM and 30 nM. In certainalternative embodiments, the dissociation constant for binding of FZD8may be between about 3 nM and 10 nM.

In certain embodiments, the FZD-binding agent or antibody binds to oneor more (for example, two or more, three or more, or four or more) humanfrizzled receptors with an EC₅₀ of about 1 μM or less, about 100 nM orless, about 40 nM or less, about 20 nM or less, about 10 nM or less, orabout 1 nM or less. For example, in certain embodiments, a FZD-bindingagent or antibody described herein that binds to more than one FZD hasan EC₅₀ of about 40 nM or less, about 20 nM or less, or about 10 nM orless, with respect to those FZDs. In certain embodiments, theFZD-binding agent or antibody has an EC⁵⁰ of about 20 nM or less withrespect to one or more (e.g., 1, 2, 3, 4, or 5) of the following FZDs:FZD1, FZD2, FZD5, FZD7, and FZD8. In certain embodiments, theFZD-binding agent or antibody has an EC₅₀ of about 10 nM or less withrespect to one or more (e.g., 1, 2, 3, 4, or 5) of the following FZDs:FZD1, FZD2, FZD5. FZD7, and FZD8. In certain embodiments, theFZD-binding agent or antibody has an EC50 of about 40 nM or less or 20nM or less with respect to binding of FZD5 and/or FZD8.

In certain embodiments, the FZD-binding agent (e.g., antibody) binds tothe same epitope as or binds to an epitope that overlaps with theepitope of an antibody comprising a heavy chain variable regioncomprising SEQ ID NO:10 and a light chain variable region comprising SEQID NO:12 or SEQ ID NO:14 (e.g., the 18R5 or 18R8 IgG antibody). Incertain embodiments, the FZD-binding agent or antibody binds to the sameepitope as or binds to an epitope that overlaps with the epitope of anantibody comprising a heavy chain comprising the sequence of SEQ ID NO:11 and a light chain comprising the sequence of SEQ ID NO:13 or SEQ IDNO:15. In certain embodiments, the FZD-binding agent binds to the sameepitope as or binds to an epitope that overlaps with the epitope of anantibody comprising a heavy chain variable region comprising SEQ IDNO:85 and a light chain variable region comprising SEQ ID NO:86 (e.g.the 44R24 IgG antibody).

In certain embodiments, the FZD-binding agent competes for specificbinding to a human frizzled receptor with an antibody in a competitivebinding assay, wherein the antibody comprises a heavy chain variableregion comprising SEQ ID NO:10 and a light chain variable regioncomprising SEQ ID NO:12 or SEQ ID NO: 14. In certain embodiments, theFZD-binding agent competes for specific binding to a human frizzledreceptor with an antibody comprising a heavy chain comprising thesequence of SEQ ID NO: 11 and a light chain comprising the sequence ofSEQ ID NO:13 or SEQ ID NO:15. In certain embodiments, the antibody withwhich the agent competes for specific binding to the human frizzledreceptor is an 18R5 IgG antibody. In certain alternative embodiments,the antibody is an 18R8 IgG antibody.

In certain embodiments, the FZD-binding agent competes for specificbinding to a human frizzled receptor with an antibody in a competitivebinding assay, wherein the antibody comprises a heavy chain variableregion comprising SEQ ID NO:85 and a light chain variable regioncomprising SEQ ID NO:86.

In certain embodiments, the FZD-binding agent directly competes forbinding or directly inhibits binding of Wnt on the FZD(s).

In certain embodiments, the FZD-binding agent or antibody binds to atleast part of a region of a human frizzled receptor designated by theinventors as the Biological Binding Site (BBS) (FIG. 9, Example 6). Inhuman FZD8 (SEQ ID NO:54), the BBS consists of the following: (a) aconformational epitope consisting of amino acids 72(F), 74-75(PL).78(I), 92(Y), 121-122(LM), and 129-132(WPDR (SEQ ID NO:70)) (the “cleft”of the BBS shown in FIGS. 8 and 9): (b) a region of FZD8 consisting ofthe sequence GLEVHQ (SEQ ID NO:25) (the “top edge” of the BBS shown inFIGS. 8 and 9); and (c) a region of FZD8 consisting of the sequence YGFA(SEQ ID NO:74) (the “bottom edge” of the BBS shown in FIGS. 8 and 9).The corresponding residues of the BBS on FZD1-7, FZD9, and FZD10 areidentified in Table 1, below, and FIG. 8. In certain embodiments, anagent that blocks binding of a ligand (e.g., a Wnt) to the FZD inhibitsthe binding of the ligand to the BBS. It is understood that in certainembodiments, the agents which bind to at least part of the BBS may alsobind one or more regions elsewhere (i.e., outside of the BBS) on thehuman frizzled receptor. In other words, in certain embodiments, theepitope to which the FZD-binding agent or antibody binds is a regionwithin the extracellular domain of the FZD receptor that overlaps withthe BBS, but is not entirely contained within the BBS. In certainalternative embodiments, the epitope to which the FZD-binding agent orantibody binds is entirely contained within the BBS (i.e., the BBScomprises the entire epitope to which the FZD binding antibody or otheragent binds).

TABLE 1 Biological Binding Sites (BBS) of FZD Receptors Human FrizzledReceptor (aa Amino acid residues forming the sequence)Biological Binding Site (BBS) FZD1147-153(GLEVHQF), 155-156(PL), 159(V), (SEQ ID173(Y), 201-202(LM), 205-212(FGFQWPDT) NO: 26) FZD270-76(GLEVHQF), 78-79(PL), 82(V), 96(Y), (SEQ ID124-125(LM), 128-135(FGFQWPER) NO: 30) FZD359-65(ALAMEPF), 67-68(PM), 71(L), 85(Y), (SEQ ID113-114(LM), 117-124(FGVPWPED) NO: 34) FZD479-85(ELQLTTF), 87-88(PL), 91(Y), 105(Y), (SEQ ID134-135(VL), 138-145(FGFAWPES) NO: 38) FZD564-70(GLEVHQF), 72-73(PL), 76(I) 90(Y), (SEQ ID119-120(LM), 123-130(YGFAWPER) NO: 42) FZD655-61(AVEMEHF), 63-64(PL), 67(L), 81(F), (SEQ ID109-110(LI), 113-120(FGIRWPEE) NO: 46) FZD780-86(GLEVHQF), 88-89(PL), 92(V), 106(Y), (SEQ ID134-135(LM), 138-145(FGFQWPER) NO: 50) FZD866-72(GLEVHQF), 74-75(PL), 78(I), 92(Y), (SEQ ID121-122(LM), and 125-132(YGFAWPDR) NO: 54) FZD970-76(AAELAEF), 78-79(PL), 82(Y), 96(Y), (SEQ ID125-126(IM), 129-136(FNFGWPDS) NO: 58) FZD1065-71(AIQLHEF), 73-74(PL), 77(Y), 91(Y), (SEQ ID120-121(IM), 124-131(FNFKWPDS) NO: 62)

Without being bound by theory, the BBS is believed to comprise apossible ligand binding site, such as a binding site for Wnt. On FZD8,this possible ligand binding site comprises the conformational epitopeformed by amino acids 72(F), 74-75(PL), 78(I), 92(Y), 121-122(LM), and129-132(WPDR (SEQ ID NO:70)) (the “cleft” of the BBS shown in FIGS. 8and 9). The corresponding residues of the possible ligand binding siteon FZD1-7, FZD9, and FZD10 are shown in the alignment of sequences inFIG. 8. In certain embodiments, an agent that blocks binding of a ligand(e.g, a Wnt) to the BBS inhibits the binding of the ligand to thisconformational epitope. It is understood that in certain embodiments,the agents which bind to at least a part of this ligand binding site mayalso bind to a region elsewhere (e.g., outside of the BBS) on the humanfrizzled receptor. In certain alternative embodiments, the agent doesnot bind to any portion of the FZD outside of the conformationalepitope.

In certain embodiments, the agent binds to at least part of the sequenceQDEAGLEVHQFWPL (SEQ ID NO:67) within the human frizzled receptor if thehuman frizzled receptor is FZD8, or the corresponding sequence if thehuman frizzled receptor is FZD1-7, FZD9, or FZD10. This region on theFZDs comprises the “top edge” of the BBS identified in FIGS. 8 and 9.The sequences corresponding to the epitope QDEAGLEVHQFWPL (SEQ ID NO:67)of FZD8 in the various frizzled receptors are identified in Table 2,below, and are also apparent from the alignment in FIG. 8. In certainembodiments, the agent specifically binds to a human frizzled receptorselected from the group consisting of FZD1, FZD2, FZD5, FZD7, and FZD8,and the agent binds to at least part of the sequenceQ(DE/ED)AGLEVHQF(Y/W)PL (SEQ ID NO:24) within the human frizzledreceptor. In certain embodiments, the agent specifically binds to atleast part of the sequence AGLEVHQF (SEQ ID NO:68) within the humanfrizzled receptor(s) FZD1, FZD2, FZD5, FZD7, and/or FZD8. In certainembodiments, the agent binds to at least part of a sequence in FZD3,FZD4, FZD6, FZD9, and/or FZD10 that corresponds to the sequence AGLEVHQF(SEQ ID NO:68) in FZD8. In certain embodiments, the agent specificallybinds to at least part of the sequence GLEVHQ (SEQ ID NO: 25) within thehuman frizzled receptor(s) FZD1, FZD2, FZD5, FZD7, and/or FZD8. Thissequence is the “top edge” of the BBS shown in FIG. 9. In certainembodiments, the agent binds to at least part of the sequence in FZD3,FZD4, FZD6, FZD9, and/or FZD10 that corresponds to the sequence GLEVHQ(SEQ ID NO: 25) in FZD8. Sequences which correspond to the sequenceGLEVHQ (SEQ ID NO: 25) of FZD8 are underlined in the second and thirdcolumns of Table 2 below and are apparent from the sequence alignment inFIG. 8. In certain embodiments, an agent that binds to at least part ofSEQ ID NO:67 or 68 in FZD8, or to a corresponding epitope in anotherFZD, inhibits binding of a ligand (e.g., a Wnt) to the FZD (e.g., to theBBS of the FZD). In certain embodiments, the agents which bind to theabove indicated regions may also bind to additional regions elsewhere(i.e., outside of the above-specified regions) on the human frizzledreceptor.

TABLE 2 Corresponding regions on human frizzled receptorsaa corresponding to aa corresponding to aa 62-75 aa 65-72aa corresponding to (QDEAGLEVHQFWPL; (AGLEVHQF; SEQ aa 124-128SEQ ID NO: 67) of ID NO: 68) of (QYGFA; SEQ ID FZDFZD8 (SEQ ID NO: 54)^(a) FZD8 (SEQ ID NO: 66) of (aa seq) NO: 54)^(a)NO: 54)^(b) FZD8 (SEQ HD FZD1 143-156 146-153 204-208 (KFGFQ)(SEQ ID NO: 26) (QEDAGLEVHQFYPL) (AGLEVHQF) FZD2 66-79 69-76 (AGLEVHQF)127-131 (KFGFQ) (SEQ ID NO: 30) (QEDAGLEVHQFYPL) FZD3 55-6858-65 (AALAMEPF) 116-120 (MFGVP) (SEQ ID NO: 34) (QQTAALAMEPFHPM) FZD475-88 78-85 (AELQLTTF) 137-141 (EFGFA) (SEQ ID NO: 38) (QTDAELQLTTFTPL)FZD5 60-73 63-70 (AGLEVHQF) 122-126 (QYGFA) (SEQ ID NO: 42)(QDEAGLEVHQFWPL) FZD6 51-64 54-61 (AAVEMEHF) 112-116 (TFGIR)(SEQ ID NO: 46) (QSIAAVEMEHFLPL) FZD7 76-89 79-86 (AGLEVHQF)137-141 (KFGFQ) (SEQ ID NO: 50) (QEDAGLEVHQFYPL) FZD9 66-7969-76 (AAAELAEF) 128-132 (QFNFG) (SEQ ID NO: 58) (QGEAAAELAEFAPL) FZD1061-74 64-71 (AAIQLHEF) 123-127 (QFNFK) (SEQ ID NO: 62) (QREAAIQLHEFAPL)^(a)Sequences corresponding to as 66-71 GLEVHQ (SEQ ID NO: 25) of FZD8(SEq ID NO: 54) are underlined. ^(b)Sequences corresponding to as125-128 YGFA (SEQ ID NO: 74) of FZD8 (SEQ ID NO: 54) are underlined.

In certain embodiments, the FZD-binding agent binds to at least part ofa region consisting of the sequence QYGFA (SEQ ID NO:66) if the humanfrizzled receptor is FZD8, which comprises the “bottom edge” of the BBS,or the corresponding sequence if the human frizzled receptor is FZD1-7,FZD9, or FZD10. The sequences corresponding to the region QYGFA (SEQ IDNO:66) in the various frizzled receptors are identified in FIG. 8 andTable 2 above. In certain embodiments, the FZD-binding agent binds to atleast part of a region consisting of the sequence YGFA (SEQ ID NO:74),the “bottom edge” of the BBS, if the human frizzled receptor is FZD8, orthe corresponding sequence if the human frizzled receptor is FZD1-7,FZD9, or FZD10. The sequences corresponding to the region YGFA (SEQ IDNO:74) in the various frizzled receptors are identified in FIG. 8 andunderlined in the fourth column of Table 2 above. In certainembodiments, an agent that binds to at least part of SEQ ID NO:66 or SEQID NO:74 in FZD8, or to its corresponding sequence in another FZD,inhibits binding of a ligand (e.g., a Wnt) to the FZD (e.g., the BBS ofthe FZD). In certain embodiments, the agents which bind to this regionmay also bind to one or more amino acid residues elsewhere (i.e. outsideof this region) on the human frizzled receptor.

In certain embodiments, FZD-binding agent binds at least part of theregion forming the “top edge” of the BBS, as well as at least a part ofthe region forming the “bottom edge” of the BBS. In certain embodiments,the FZD-binding agent that binds to at least part ofQ(DE/ED)AGLEVHQF(Y/W)PL (SEQ ID NO:24) within FZD1, FZD2, FZD5, FZD7,and/or FZD8, QDEAGLEVHQFWPL (SEQ ID NO:67) within FZD8, AGLEVHQF (SEQ IDNO:68) within FZD8, and/or GLEVHQ (SEQ ID NO:25) within FZD8, and/or asequence corresponding to any of these sequences in a different humanfrizzled receptor (as defined in Table 2, above) further binds to atleast part of QYGFA (SEQ ID NO:66) within FZD8 or YGFA (SEQ ID NO:74)within FZD8, and/or a sequence corresponding to one of these sequenceswithin FZD1-7, FZD9, or FZD10 (as defined in Table 2, above). In certainembodiments, the FZD binding agent binds to at least part of thesequence GLEVHQ (SEQ ID NO:25) within FZD8, as well as to at least partof the sequence YGFA (SEQ ID NO:74) within FZD8. In certain embodiments,the FZD-binding agent binds to at least part of a region of FZD1-7,FZD9, or FZD10 corresponding to the sequence GLEVHQ (SEQ ID NO:25) inFZD8, as well as to at least part of a region of FZD1-7, FZD9, or FZD10corresponding to the sequence YGFA (SEQ ID NO:74) in FZD8. In certainembodiments, the FZD-binding agent that binds to the indicated sequencesalso binds to one or more sequences elsewhere within the human frizzledreceptor(s) to which it binds. In other words, in certain embodiments,the epitope to which the FZD-binding agent or antibody binds is a regionwithin the FZD extracellular domain that overlaps only partially withthe above-indicated sequences. In certain alternative embodiments, theentire epitope to which the FZD-binding agent binds is entirelycontained within the above-indicated sequences.

In certain embodiments, the FZD-binding agent binds a human FZD receptor(e.g., FZD8) in the cleft of its BBS. See, e.g., the cleft residueslisted in Table 16 in the Examples below. In certain embodiments, L75 ofFZD8 is necessary for binding of the agent. In certain embodiments, theFZD-binding agent which binds in the cleft of the BBS, may have amolecular weight of less than about 2000 Daltons or less than about 1000Daltons. The FZD-binding agent may be a polypeptide, a lipid, anaptamer, a small molecule or other compound. In certain embodiments, theFZD-binding agent comprises a polypeptide comprising about 100 aminoacids or less. In certain embodiments, the polypeptide comprises lessthan about 20 amino acids. The polypeptide may be modified orunmodified. In certain embodiments, the agent comprises a polypeptidecomprising a sequence having at least about 80% sequence identity to asequence selected from the group consisting of SEQ ID NOs: 98-111 and113-116. In certain embodiments, the agent comprises SEQ ID NO:112. Insome instances, the agent may comprise SEQ ID NO: 108.

In certain embodiments, the agent is a polypeptide. In certainembodiments, the agent or polypeptide is an antibody. In certainembodiments, the antibody is an IgG1 antibody or an IgG2 antibody. Incertain embodiments, the antibody is a monoclonal antibody. In certainembodiments, the antibody is a human antibody or a humanized antibody.In certain embodiments, the antibody is an antibody fragment.

The antibodies or other agents of the present invention can be assayedfor specific binding by any method known in the art. The immunoassayswhich can be used include, but are not limited to competitive andnon-competitive assay systems using techniques such as BIAcore analysis.FACS analysis, immunofluorescence, immunocytochemistry, Western blots,radioimmunoassays, ELISA, “sandwich” immunoassays, immunoprecipitationassays, precipitation reactions, gel diffusion precipitin reactions,immunodiffusion assays, agglutination assays, complement-fixationassays, immunoradiometric assays, fluorescent immunoassays, and proteinA immunoassays. Such assays are routine and well known in the art (see,e.g., Ausubel et al, eds. 1994, Current Protocols in Molecular Biology,Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated byreference herein in its entirety).

For example, the specific binding of an antibody to a human frizzledreceptor may be determined using ELISA. An ELISA assay comprisespreparing antigen, coating wells of a 96 well microtiter plate withantigen, adding the FZD-binding antibody or other FZD-binding agentconjugated to a detectable compound such as an enzymatic substrate (e.g.horseradish peroxidase or alkaline phosphatase) to the well, incubatingfor a period of time and detecting the presence of the antigen. In someembodiments, the FZD-binding antibody or agent is not conjugated to adetectable compound, but instead a second conjugated antibody thatrecognizes the FZD-binding antibody or agent is added to the well. Insome embodiments, instead of coating the well with the antigen, theFZD-binding antibody or agent can be coated to the well and a secondantibody conjugated to a detectable compound can be added following theaddition of the antigen to the coated well. One of skill in the artwould be knowledgeable as to the parameters that can be modified toincrease the signal detected as well as other variations of ELISAs knownin the art (see e.g. Ausubel et al, eds, 1994, Current Protocols inMolecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1).

The binding affinity of an antibody or other agent to a human frizzledreceptor and the off-rate of an antibody-antigen interaction can bedetermined by competitive binding assays. One example of a competitivebinding assay is a radioimmunoassay comprising the incubation of labeledantigen (e.g. ³H or ¹²⁵I), or fragment or variant thereof, with theantibody of interest in the presence of increasing amounts of unlabeledantigen followed by the detection of the antibody bound to the labeledantigen. The affinity of the antibody against a frizzled receptor andthe binding off-rates can be determined from the data by scatchard plotanalysis. In some embodiments, BIAcore kinetic analysis is used todetermine the binding on and off rates of antibodies or agents that bindone or more human frizzled receptors. BIAcore kinetic analysis comprisesanalyzing the binding and dissociation of antibodies from chips withimmobilized FZD antigens on their surface.

In certain embodiments, the agent (e.g. antibody) is an antagonist of atleast one human frizzled receptor (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 FZDs) bound by the agent. In certain embodiments, the agent inhibitsat least about 10%, at least about 20%, at least about 30%, at leastabout 50%, at least about 75%, at least about 90%, or about 100% of oneor more activity of the bound human frizzled receptor.

In certain embodiments, the FZD-binding agent inhibits binding of aligand to the at least one human frizzled receptor. In certainembodiments, the FZD-binding agent inhibits binding of a ligand to theBiological Binding site (BBS) of the human frizzled receptor. In certainembodiments, the ligand is a human Wnt protein. Nineteen human Wntproteins have been identified: WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4,WNT5A, WNT5B, WNT6. WNT7A, WNT7B. WNT8A, WNT8B. WNT9A (previouslyWNT14), WNT9B (previously WNT15), WNT10A, WNT10B, WNT11, and WNT16. Incertain embodiments, the agent inhibits binding of WNT3A to FZD8. Incertain embodiments, the inhibition of binding of a particular ligand toa particular human frizzled protein provided by the FZD-binding agent isat least about 10%, at least about 25%, at least about 50%, at leastabout 75%, at least about 90%, or at least about 95%. In certainembodiments, an agent that inhibits binding of a ligand such as a Wnt toa FZD, further inhibits Wnt signaling (e.g., inhibits canonical Wntsignaling).

In certain embodiments, the FZD-binding agent inhibits Wnt signaling. Itis understood that a FZD-binding agent that inhibits Wnt signaling may,in certain embodiments, inhibit signaling by one or more Wnts, but notnecessarily by all Wnts. In certain alternative embodiments, signalingby all human Wnts may be inhibited. In certain embodiments, signaling byone or more Wnts selected from the group consisting of WNT1, WNT2,WNT2B/13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A,WNT8B, WNT9A (previously WNT14), WNT9B (previously WNT15), WNT10A,WNT10B, WNT11, and WNT16 is inhibited. In certain embodiments, the Wntsignaling that is inhibited is signaling by WNT1, WNT2. WNT3, WNT3A,WNT7a, WNT7b, and/or WNT10B. In certain embodiments, the agent inhibitssignaling by (at least) WNT1, WNT3A, WNT7b, and WNT10B. In particularembodiments, the agent inhibits signaling by (at least) WNT3A. Incertain embodiments, the inhibition of signaling by a Wnt provided bythe FZD-binding agent is a reduction in the level of signaling by theWnt of least about 10%, at least about 25%, at least about 50%, at leastabout 75%, at least about 90%, or at least about 95%. In certainembodiments, the Wnt signaling that is inhibited is canonical Wntsignaling.

In vivo and in vitro assays for determining whether a FZD-binding agent(or candidate FZD-binding agent) inhibits Wnt signaling are known in theart. For example, cell-based, luciferase reporter assays utilizing aTCF/Luc reporter vector containing multiple copies of the TCF-bindingdomain upstream of a firefly luciferase reporter gene may be used tomeasure canonical Wnt signaling levels in vitro (Gazit et al., 1999,Oncogene 18; 5959-66). The level of Wnt signaling in the presence of oneor more Wnts (e.g., Wnt(s) expressed by transfected cells or provided byWnt-conditioned media) with the FZD-binding agent present is compared tothe level of signaling without the FZD-binding agent present.Non-limiting, specific examples of the use of such a luciferase reporterassay to assess inhibition of canonical Wnt signaling are provided inExamples 3 and 11, below. In addition to the TCF/luc reporter assay, theeffect of a FZD-binding agent (or candidate agent) on canonical Wntsignaling may be measured in vitro or in vivo by measuring the effect ofthe agent on the level of expression of beta-catenin regulated genes,such as c-myc (He et al., Science. 281:1509-12 (1998)), cyclin D1 (Tetsuet al., Nature, 398:422-6 (1999)) and/or fibronectin (Gradl et al. Mol.Cell. Biol., 19:5576-87 (1999)). In certain embodiments, the effect ofan agent on Wnt signaling may also be assessed by measuring the effectof the agent on the phosphorylation state of Dishevelled-1,Dishevelled-2, Dishevelled-3, LRP5, LRP6, and/or beta-catenin. In stillfurther embodiments, the effect of a FZD-binding agent on Wnt signalingis determined by assessing the impact of the FZD-binding agent on theexpression level of one or more genes in a Wnt signature.

In certain embodiments, the FZD-binding agents have one or more of thefollowing effects: inhibit proliferation of tumor cells, reduce thetumorigenicity of a tumor by reducing the frequency of cancer stem cellsin the tumor, inhibit tumor growth, increase survival, trigger celldeath of tumor cells, differentiate tumorigenic cells to anon-tumorigenic state, or prevent metastasis of tumor cells.

In certain embodiments, antibodies or other agents that specificallybind one or more human frizzled receptors trigger cell death via aconjugated toxin, chemotherapeutic agent, radioisotope, or other suchagent. For example, in certain embodiments, an antibody to a humanfrizzled antibody is conjugated to a toxin that is activated in tumorcells expressing the FZD by protein internalization. In certainalternative embodiments, the agent or antibody is not conjugated to atoxin, chemotherapeutic agent, or radioisotope.

In certain embodiments, the FZD-binding agents are capable of inhibitingtumor growth. In certain embodiments, the FZD-binding agents are capableof inhibiting tumor growth in vivo (e.g., in a xenograft mouse modeland/or in a human having cancer).

In certain embodiments, the FZD-binding agents are capable of reducingthe tumorigenicity of a tumor. In certain embodiments, the agent orantibody is capable of reducing the tumorigenicity of a tumor comprisingcancer stem cells in an animal model, such as a mouse xenograft model.In certain embodiments, the number or frequency of cancer stem cells ina tumor is reduced by at least about two-fold, about three-fold, aboutfive-fold, about ten-fold, about 50-fold, about 100-fold, or about1000-fold. In certain embodiments, the reduction in the number orfrequency of cancer stem cells is determined by limiting dilution assayusing an animal model. An example of a limiting dilution assay used totest the efficacy of an anti-FZD antibody is provided in Example 8,below. Additional examples and guidance regarding the use of limitingdilution assays to determine a reduction in the number or frequency ofcancer stem cells in a tumor can be found, e.g., in InternationalPublication Number WO 2008/042236, U.S. Patent Application PublicationNo. 2008/0064049, and U.S. Patent Application Publication No.2008/0178305, each of which is incorporated by reference herein in itsentirety.

In certain embodiments, antibodies to human frizzled receptors mediatecell death of a cell expressing the FZD protein via antibody-dependentcellular cytotoxicity (ADCC). ADCC involves cell lysis by effector cellsthat recognize the Fec portion of an antibody. Many lymphocytes,monocytes, tissue macrophages, granulocytes and eosinophiles, forexample, have Fc receptors and can mediate cytolysis (Dillman, 1994. J.Clin. Oncol. 12:1497).

In certain embodiments, antibodies to one or more FZDs trigger celldeath of a cell expressing the FZD protein(s) by activatingcomplement-dependent cytotoxicity (CDC). CDC involves binding of serumcomplement to the Fc portion of an antibody and subsequent activation ofthe complement protein cascade, resulting in cell membrane damage andeventual cell death. Biological activity of antibodies is known to bedetermined, to a large extent, by the constant domains or Fc region ofthe antibody molecule (Uananue and Benacerraf, Textbook of Immunology,2nd Edition. Williams & Wilkins, p. 218 (1984)). Antibodies of differentclasses and subclasses differ in this respect, as do antibodies of thesame subclass but from different species. Of human antibodies. 1 μM isthe most efficient class of antibodies to bind complement, followed byIgG1, IgG3, and IgG2 whereas IgG4 appears quite deficient in activatingthe complement cascade (Dillman, 1994, J. Clin. Oncol. 12:1497; Jefferiset al., 1998, Immunol. Rev. 163:59-76). According to the presentinvention, antibodies of those classes having the desired biologicalactivity are prepared.

The ability of any particular antibody against one or more FZDs tomediate lysis of the target cell by complement activation and/or ADCCcan be assayed. The cells of interest are grown and labeled in vitro;the antibody is added to the cell culture in combination with eitherserum complement or immune cells which can be activated by the antigenantibody complexes. Cytolysis of the target cells is detected, forexample, by the release of label from the lysed cells. In fact,antibodies can be screened using the patient's own serum as a source ofcomplement and/or immune cells. The antibody that is capable ofactivating complement or mediating ADCC in the in vitro test can then beused therapeutically in that particular patient.

The invention provides polypeptides, including, but not limited to,antibodies that specifically bind to one or more human frizzledreceptors, that comprise one, two, three, four, five and/or six of theCDRs of 18R5 and/or 18R8 (see Table 4 of Example 1 below) with up tofour (i.e., 0, 1, 2, 3, or 4) conservative amino acid substitutions perCDR. Thus, the invention provides polypeptides, including, but notlimited to, antibodies that specifically bind to one or more humanfrizzled receptors that comprise one, two, three, four, five and/or sixof the CDRs of 18R5 and/or 18R8. In certain embodiments, thepolypeptides comprise the heavy chain CDR3 of 18R8 and/or the lightchain CDR3 of 18R5 or 18R8. In certain embodiments, the heavy chainCDR(s) are contained within a heavy chain variable region and/or thelight chain CDR(s) are contained within a light chain variable region.

For example, the invention provides a polypeptide (e.g., an antibody)that specifically binds a human frizzled receptor, wherein thepolypeptide comprises a heavy chain variable region comprising: (a) aheavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO:1), or a variantthereof comprising 1, 2, 3, or 4 amino acid substitutions; (b) a heavychain CDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO:2), or a variantthereof comprising 1, 2, 3, or 4 amino acid substitutions; and/or (c) aheavy chain CDR3 comprising NFIKYVFAN (SEQ ID NO:3), or a variantthereof comprising 1, 2, 3, or 4 amino acid substitutions. In certainembodiments, the polypeptide further comprises a light chain variableregion comprising: (a) a light chain CDR1 comprising SGDKLGKKYAS (SEQ IDNO:4) or SGDNIGSFYVH (SEQ ID NO:7), or a variant of SEQ ID NO:4 or SEQID NO:7 comprising 1, 2, 3, or 4 amino acid substitutions; (b) a lightchain CDR2 comprising EKDNRPSG (SEQ ID NO:5) or DKSNRPSG (SEQ ID NO:8),or a variant of SEQ ID NO:5 or SEQ ID NO:8 comprising 1, 2, 3, or 4amino acid substitutions; and/or (c) a light chain CDR3 comprisingSSFAGNSLE (SEQ ID NO:6) or QSYANTLSL (SEQ ID NO:9), or a variant of SEQID NO:6 or SEQ ID NO:9 comprising 1, 2, 3, or 4 amino acidsubstitutions. In certain embodiments, the amino acid substitutions areconservative substitutions.

Thus, the invention provides polypeptides or antibodies that comprise aheavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO:1), a heavy chain CDR2comprising VISGDGSYTYYADSVKG (SEQ ID NO:2), and/or a heavy chain CDR3comprising NFIKYVFAN (SEQ ID NO:3). In certain embodiments, the lightchain CDR(s) are contained within a variable region of an antibody heavychain. In certain embodiments, the polypeptide or antibody comprisingthe one or more of heavy chain CDRs specifically binds one or more humanfrizzled receptors. In certain embodiments, the CDR(s) have beenmodified with 1, 2, 3, or 4 conservative amino acid substitutions. Incertain embodiments, each of the heavy chain CDR(s) has been modified byno more than 1-2 conservative amino acid substitutions.

The invention also provides an antibody that specifically binds a humanfrizzled receptor, wherein the antibody comprises a heavy chain variableregion comprising: (a) a heavy chain CDR I comprising GFTFSHYTLS (SEQ IDNO:1), or a variant thereof comprising 1, 2, 3, or 4 amino acidsubstitutions: (b) a heavy chain CDR2 comprising VISGDGSYTYYADSVKG (SEQID NO:2), or a variant thereof comprising 1, 2, 3, or 4 amino acidsubstitutions; and (c) a heavy chain CDR3 comprising NFIKYVFAN (SEQ IDNO:3), or a variant thereof comprising 1, 2, 3, or 4 amino acidsubstitutions. In certain embodiments, the antibody further comprises alight chain variable region comprising: (a) a light chain CDR1comprising SGDKLGKKYAS (SEQ ID NO:4) or SGDNIGSFYVH (SEQ ID NO:7), or avariant of SEQ ID NO:4 or SEQ ID NO:7 comprising 1, 2, 3, or 4 aminoacid substitutions; (b) a light chain CDR2 comprising EKDNRPSG (SEQ IDNO:5) or DKSNRPSG (SEQ ID NO:8), or a variant of SEQ ID NO:5 or SEQ IDNO:8 comprising 1, 2, 3, or 4 amino acid substitutions; and (c) a lightchain CDR3 comprising SSFAGNSLE (SEQ ID NO:6) or QSYANTLSL (SEQ IDNO:9), or a variant of SEQ ID NO:6 or SEQ ID NO:9 comprising 1, 2, 3, or4 amino acid substitutions. In some alternative embodiments, theantibody instead further comprises a light chain variable regioncomprising: (a) a light chain CDR1 comprising SGDKLGKKYAS (SEQ ID NO:4)or SGDNIGSFYVH (SEQ ID NO:7): (b) a light chain CDR2 comprising EKDNRPSG(SEQ ID NO:5) or DKSNRPSG (SEQ ID NO:8); and (c) a light chain CDR3comprising SSFAGNSLE (SEQ ID NO:6) or QSYANTLSL (SEQ ID NO:9). Incertain embodiments, the antibody specifically binds FZD1, FZD2, FZD5,FZD7, and/or FZD8. In certain embodiments, the antibody specificallybinds two or more human frizzled receptors including FZD5 and FZD8. Incertain embodiments, the amino acid substitutions are conservativesubstitutions.

The invention further provides a polypeptide (e.g., an antibody) thatspecifically binds a human frizzled receptor, wherein the polypeptidecomprises a light chain variable region comprising: (a) a light chainCDR1 comprising SGDKLGKKYAS (SEQ ID NO:4) or SGDNIGSFYVH (SEQ ID NO:7),or a variant of SEQ ID NO:4 or SEQ ID NO:7 comprising 1, 2, 3, or 4amino acid substitutions; (b) a light chain CDR2 comprising EKDNRPSG(SEQ ID NO:5) or DKSNRPSG (SEQ ID NO:8), or a variant of SEQ ID NO:5 orSEQ ID NO:8 comprising 1, 2, 3, or 4 amino acid substitutions: and/or(c) a light chain CDR3 comprising SSFAGNSLE (SEQ ID NO:6) or QSYANTLSL(SEQ ID NO:9), or a variant of SEQ ID NO:6 or SEQ ID NO:9 comprising 1,2, 3, or 4 amino acid substitutions. In certain embodiments, the aminoacid substitutions are conservative substitutions.

Also provided are polypeptides or antibodies that comprise (a) a lightchain CDR1 comprising the sequence SGD(K/N)(L/I)G(K/S)(K/F)Y(A/V)(S/H)(SEQ ID NO:71) or the sequence of SEQ ID NO:71 with up to four (i.e., 0,1, 2, 3, or 4) conservative amino acid substitutions, (b) a light chainCDR2 comprising the sequence (E/D)K(D/S)NRPSG (SEQ ID NO:72) or thesequence of SEQ ID NO:72 with up to four conservative amino acidsubstitutions, and/or (c) a light chain CDR3 comprising the sequence(S/Q)S(F/Y)A(G/N)(N/T)(no aa/L)SL(E/no aa) (where “no aaL” indicateseither L or no amino acid and “E/no aa” indicates either E or no aminoacid; SEQ NO:73) or the sequence of SEQ ID NO:73 with up to fourconservative amino acid substitutions.

The invention also provides polypeptides or antibodies that comprise alight chain CDR1 comprising SGDKLGKKYAS (SEQ ID NO:4) or SGDNIGSFYVH(SEQ ID NO:7), a light chain CDR2 comprising EKDNRPSG (SEQ ID NO:5) orDKSNRPSG (SEQ ID NO:8), and/or a light chain CDR3 comprising SSFAGNSLE(SEQ ID NO:6) or QSYANTLSL (SEQ ID NO:9). In certain embodiments, thepolypeptide or antibody comprises a light chain CDR1 comprisingSGDNIGSFYVH (SEQ ID NO:7), a light chain CDR2 comprising DKSNRPSG (SEQID NO:8), and a light chain CDR3 comprising QSYANTLSL (SEQ ID NO:9). Incertain alternative embodiments, the polypeptide or antibody comprises alight chain CDR1 comprising SGDKLGKKYAS (SEQ ID NO:4), a light chainCDR2 comprising EKDNRPSG (SEQ ID NO:5), and a light chain CDR3comprising SSFAGNSLE (SEQ ID NO:6). In certain embodiments, the lightchain CDR(s) are contained within a variable region of an antibody lightchain. In certain embodiments, the polypeptide or antibody specificallybinds one or more human frizzled receptors. In certain embodiments, thepolypeptide or antibody comprising the one or more of light chain CDRsspecifically binds one or more human frizzled receptors. In certainembodiments, the CDR(s) have been modified with 1, 2, 3, or 4conservative modifications. In certain embodiments, each of the lightchain CDR(s) has been modified by no more than 1-2 conservative aminoacid substitutions.

In certain embodiments, the antibody comprises (a) a heavy chain CDR1comprising GFTFSHYTLS (SEQ ID NO:1), a heavy chain CDR2 comprisingVISGDGSYTYYADSVKG (SEQ ID NO:2), and a heavy chain CDR3 comprisingNFTKYVFAN (SEQ ID NO:3); and/or (b) a light chain CDR1 comprisingSGDKLGKKYAS (SEQ ID NO:4) or SGDNIGSFYVH (SEQ ID NO:7), a light chainCDR2 comprising EKDNRPSG (SEQ ID NO:5) or DKSNRPSG (SEQ ID NO:8), and alight chain CDR3 comprising SSFAGNSLE (SEQ ID NO:6) or QSYANTLSL (SEQ IDNO:9). In certain embodiments, the antibody comprises (a) a heavy chainCDR1 comprising GFTFSHYTLS (SEQ ID NO:1), a heavy chain CDR2 comprisingVISGDGSYTYYADSVKG (SEQ ID NO:2), and a heavy chain CDR3 comprisingNFIKYVFAN (SEQ ID NO:3); and (b) a light chain CDR1 comprisingSGDKLGKKYAS (SEQ ID NO:4), a light chain CDR2 comprising EKDNRPSG (SEQID NO:5), and a light chain CDR3 comprising SSFAGNSLE (SEQ ID NO:6). Incertain embodiments, the antibody comprises (a) a heavy chain CDR1comprising GFTFSHYTLS (SEQ ID NO: 1), a heavy chain CDR2 comprisingVISGDGSYTYYADSVKG (SEQ ID NO:2), and a heavy chain CDR3 comprisingNFIKYVFAN (SEQ ID NO:3) and (b) a light chain CDR1 comprisingSGDNIGSFYVH (SEQ ID NO:7), a light chain CDR2 comprising DKSNRPSG (SEQID NO:8), and a light chain CDR3 comprising QSYANTLSL (SEQ ID NO:9). Incertain embodiments, the CDR(s) have been modified with 1, 2, 3, or 4conservative amino acid substitutions. In certain embodiments, each ofthe CDR(s) has been modified by no more than 1-2 conservative amino acidsubstitutions.

The invention further provides polypeptides, including, but not limitedto, antibodies that specifically bind to one or more human frizzledreceptors, that comprise one, two, three, four, five and/or six of theCDRs of the anti-FZD antibody 44R24 (see Table 7 of Example 18 below)with up to four (i.e., 0, 1, 2, 3, or 4) conservative amino acidsubstitutions per CDR. Thus, the invention provides polypeptides,including, but not limited to, antibodies that specifically bind to oneor more human frizzled receptors that comprise one, two, three, four,five and/or six of the CDRs of 44R24. In certain embodiments, thepolypeptides comprise the heavy chain CDR3 of 44R24 and/or the lightchain CDR3 of 44R24. In certain embodiments, the heavy chain CDR(s) arecontained within a heavy chain variable region and/or the light chainCDR(s) are contained within a light chain variable region.

The invention also provides a polypeptide (e.g. an antibody) thatspecifically binds human FZD5 and/or FZD8, wherein the antibodycomprises: (a) a heavy chain CDR1 comprising GFTFSSYYIT (SEQ ID NO:77),or a variant thereof comprising 1, 2, 3, or 4 conservative amino acidsubstitutions; a heavy chain CDR2 comprising TISYSSSNTYYADSVKG (SEQ IDNO:78), or a variant thereof comprising 1, 2, 3, or 4 conservative aminoacid substitutions; and a heavy chain CDR3 comprising SIVFDY (SEQ IDNO:79), or a variant thereof comprising 1, 2, 3, or 4 conservative aminoacid substitutions; and/or (b) a light chain CDR1 comprising SGDALGNRYVY(SEQ ID NO:80), or a variant thereof comprising 1, 2, 3, or 4conservative amino acid substitutions; a light chain CDR2 comprising SG(SEQ ID NO:81), or a variant thereof comprising 1, 2, 3, or 4conservative amino acid substitutions; and a light chain CDR3 comprisingGSWDTRPYPKY (SEQ ID NO:82), or a variant thereof comprising 1, 2, 3, or4 conservative amino acid substitutions. In certain embodiments, theantibody (or other FZD-binding polypeptide) comprises: (a) a heavy chainCDR1 comprising GFTFSSYYIT (SEQ ID NO:77), a heavy chain CDR2 comprisingTISYSSSNTYYADSVKG (SEQ ID NO:78), and a heavy chain CDR3 comprisingSIVFDY (SEQ ID NO:79); and/or (b) a light chain CDR1 comprisingSGDALGNRYVY (SEQ ID NO:80), a light chain CDR2 comprising SG (SEQ IDNO:81), and a light chain CDR3 comprising GSWDTRPYPKY (SEQ ID NO:82).

Polypeptides comprising one of the individual light chains or heavychains described herein, as well as polypeptides (e.g., antibodies)comprising both a light chain and a heavy chain are also provided.

Also provided are polypeptides that comprise: (a) a polypeptide havingat least about 80% sequence identity to SEQ ID NO: 10; and/or (b) apolypeptide having at least about 80% sequence identity to SEQ ID NO:12or SEQ ID NO:14. In certain embodiments, the polypeptide comprises apolypeptide having at least about 85%, at least about 90%, at leastabout 95%, at least about 97%, or at least about 99% sequence identityto SEQ ID NOs:10, 12, or 14. Thus, in certain embodiments, thepolypeptide comprises (a) a polypeptide having at least about 95%sequence identity to SEQ ID NO:10, and/or (b) a polypeptide having atleast about 95% sequence identity to SEQ ID NO:12 or 14. In certainembodiments, the polypeptide comprises (a) a polypeptide having theamino acid sequence of SEQ ID NO: 10; and/or (b) a polypeptide havingthe amino acid sequence of SEQ ID NO:12 or SEQ ID NO:14. In certainembodiments, the polypeptide comprises (a) a polypeptide having theamino acid sequence of SEQ ID NO: 11: and/or (b) a polypeptide havingthe amino acid sequence of SEQ ID NO:13 or SEQ ID NO:15. In certainembodiments, the polypeptide is an antibody and/or the polypeptidespecifically binds one or more human frizzled receptors (e.g., FZD1,FZD2. FZD5, FZD7 and/or FZD8). For example, the invention provides anantibody that specifically binds a human frizzled receptor thatcomprises (a) a polypeptide having the amino acid sequence of SEQ ID NO:10; and (b) a polypeptide having the amino acid sequence of SEQ ID NO:14. In certain embodiments the polypeptide comprising SEQ ID NO:10 is aheavy chain variable region. In certain embodiments, the polypeptidecomprising SEQ ID NO:12 or 14 is a light chain variable region. Incertain embodiments, the polypeptide having a certain percentage ofsequence identity to SEQ ID NO:10, 12, or 14 differs from SEQ ID NO:10,12, or 14 by conservative amino acid substitutions only.

In certain embodiments the polypeptide or antibody comprises: (a) SEQ IDNO:10 and SEQ ID NO: 12; (b) SEQ ID NO: 10 and SEQ ID NO:14; (c) SEQ IDNO:11 and SEQ ID NO:13; or (d) SEQ ID NO:11 and SEQ ID NO: 15.

The invention further provides an antibody or other polypeptide thatspecifically binds to FZD5 and/or FZD8 and comprises: (a) a polypeptidehaving at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 97%, or at least about 99% identity toSEQ ID NO:85; and/or (b) a polypeptide having at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about97%, or at least about 99% identity to SEQ ID NO:86. In certainalternative embodiments, the polypeptide or antibody comprises SEQ IDNO:85 and/or SEQ ID NO:86.

In certain embodiments, the FZD-binding agent comprises, consistsessentially of, or consists of an anti-FZD antibody selected from thegroup consisting of 18R8, 18R5, 18R4605, 18R4805, and 44R24 IgGantibodies.

In certain embodiments, the FZD-binding agent is not the 44M1, 44M2,44M13, 44M15, 44M17, 44M18, 18M4, 18M6, 18M17, 18M18, 54M1, 54M2, 54M3,54M4, 54M5, 54M6, or 54M7 antibody reported in U.S. Patent ApplicationPublication No. US 2007/0116701, which is incorporated by referenceherein in its entirety. In certain embodiments, the FZD-binding agent isan antibody that binds FZD5, but is not 44M 13.

In certain embodiments, the FZD-binding agent comprises the heavy chainsand light chains of the 18R8 IgG2 antibody (with or without the leadersequence). In certain embodiments, the FZD-binding agent is the 18R8IgG2 antibody. DNA encoding the heavy chains and light chains of the18R8 IgG2 antibody was deposited with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va., USA, underthe conditions of the Budapest Treaty on Sep. 29, 2008, and assignedATCC deposit designation number PTA-9540. In certain embodiments, theFZD-binding agent comprises the heavy chains and light chains of the18R5 IgG2 antibody (with or without the leader sequence). In certainembodiments, the FZD-binding agent is the 18R5 IgG2 antibody. DNAencoding the heavy chains and light chains of the 18R5 IgG2 antibody wasdeposited with the ATCC, under the conditions of the Budapest Treaty onSep. 29, 2008, and assigned ATCC deposit designation number PTA-9541.

In certain embodiments, the FZD-binding agent is an IgG antibody encodedby the plasmid deposited with the ATCC on Aug. 26, 2009, and assigneddeposit designation number PTA-10307. PTA-10309, or PTA-10311.

In certain embodiments, the FZD-binding agent is an agent that competesfor specific binding to FZD1, FZD2, FZD5, FZD7, and/or FZD8 with anantibody encoded by the plasmid having ATCC deposit designation numberPTA-9540, PTA-9541, PTA-10307, or PTA-10309 (e.g., in a competitivebinding assay). In certain alternative embodiments, the FZD-bindingagent is an agent that competes for specific binding to FZD5 and/or FZD8with an antibody encoded by the plasmid having ATCC deposit designationnumber PTA-10311.

In certain embodiments, the FZD-binding agent has a circulatinghalf-life in mice, cynomologous monkeys, or humans of at least about 10hours, at least about 24 hours, at least about 3 days, at least about 1week, or at least about 2 weeks. In certain embodiments, the FZD-bindingagent is an IgG (e.g., IgG1 or IgG2) antibody that has a circulatinghalf-life in mice, cynomologous monkeys, or humans of at least about 10hours, at least about 24 hours, at least about 3 days, at least about 1week, or at least about 2 weeks. Methods of increasing the half-life ofagents such as polypeptides and antibodies are known in the art. Forexample, known methods of increasing the circulating half-life of IgGantibodies include the introduction of mutations in the Fc region whichincrease the pH-dependent binding of the antibody to the neonatal Fcreceptor (FcRn) at pH 6.0 (see, e.g. U.S. Pat. Pub. Nos. 2005/0276799,2007/0148164, and 2007/0122403). Known methods of increasing thecirculating half-life of antibody fragments lacking the Fc regioninclude such techniques as PEGylation.

Polyclonal antibodies can be prepared by any known method. Polyclonalantibodies are raised by immunizing an animal (e.g. a rabbit, rat,mouse, donkey, etc) by multiple subcutaneous or intraperitonealinjections of the relevant antigen (a purified peptide fragment,full-length recombinant protein, fusion protein, etc) optionallyconjugated to keyhole limpet hemocyanin (KLH), serum albumin, etc.diluted in sterile saline and combined with an adjuvant (e.g. Completeor Incomplete Freund's Adjuvant) to form a stable emulsion. Thepolyclonal antibody is then recovered from blood, ascites and the like,of an animal so immunized. Collected blood is clotted, and the serumdecanted, clarified by centrifugation, and assayed for antibody titer.The polyclonal antibodies can be purified from serum or ascitesaccording to standard methods in the art including affinitychromatography, ion-exchange chromatography, gel electrophoresis,dialysis, etc.

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein (1975) Nature 256:495. Using thehybridoma method, a mouse, hamster, or other appropriate host animal, isimmunized as described above to elicit the production by lymphocytes ofantibodies that will specifically bind to an immunizing antigen.Lymphocytes can also be immunized in vitro. Following immunization, thelymphocytes are isolated and fused with a suitable myeloma cell lineusing, for example, polyethylene glycol, to form hybridoma cells thatcan then be selected away from unfused lymphocytes and myeloma cells.Hybridomas that produce monoclonal antibodies directed specificallyagainst a chosen antigen as determined by immunoprecipitation,immunoblotting, or by an in vitro binding assay (e.g. radioimmunoassay(RIA); enzyme-linked immunosorbent assay (ELISA)) can then be propagatedeither in vitro culture using standard methods (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, 1986) or in vivo asascites tumors in an animal. The monoclonal antibodies can then bepurified from the culture medium or ascites fluid as described forpolyclonal antibodies above.

Alternatively monoclonal antibodies can also be made using recombinantDNA methods as described in U.S. Pat. No. 4,816,567. The polynucleotidesencoding a monoclonal antibody are isolated from mature B-cells orhybridoma cell, such as by RT-PCR using oligonucleotide primers thatspecifically amplify the genes encoding the heavy and light chains ofthe antibody, and their sequence is determined using conventionalprocedures. The isolated polynucleotides encoding the heavy and lightchains are then cloned into suitable expression vectors, which whentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, monoclonal antibodies aregenerated by the host cells. Also, recombinant monoclonal antibodies orfragments thereof of the desired species can be isolated from phagedisplay libraries expressing CDRs of the desired species as described(McCafferty et al., 1990, Nature, 348:552-554; Clackson et al., 1991,Nature. 352:624-628; and Marks et al., 1991. J. Mol. Biol.,222:581-597).

The polynucleotide(s) encoding a monoclonal antibody can further bemodified in a number of different manners using recombinant DNAtechnology to generate alternative antibodies. In some embodiments, theconstant domains of the light and heavy chains of, for example, a mousemonoclonal antibody can be substituted 1) for those regions of, forexample, a human antibody to generate a chimeric antibody or 2) for anon-immunoglobulin polypeptide to generate a fusion antibody. In someembodiments, the constant regions are truncated or removed to generatethe desired antibody fragment of a monoclonal antibody. Site-directed orhigh-density mutagenesis of the variable region can be used to optimizespecificity, affinity, etc. of a monoclonal antibody.

In some embodiments, the monoclonal antibody against the human frizzledreceptor(s) is a humanized antibody. In certain embodiments, suchantibodies are used therapeutically to reduce antigenicity and HAMA(human anti-mouse antibody) responses when administered to a humansubject. Humanized antibodies can be produced using various techniquesknown in the art. In certain alternative embodiments, the antibody tothe human frizzled receptor(s) is a human antibody. In certainembodiments, the agent is an antibody, such as a monoclonal antibody,that is not a murine antibody.

Human antibodies can be directly prepared using various techniques knownin the art. Immortalized human B lymphocytes immunized in vitro orisolated from an immunized individual that produce an antibody directedagainst a target antigen can be generated (See, e.g., Cole et al.,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985);Boerner et al., 1991, J. Immunol., 147 (1):86-95; and U.S. Pat. No.5,750,373). Also, the human antibody can be selected from a phagelibrary, where that phage library expresses human antibodies, asdescribed, for example, in Vaughan et al., 1996, Nat. Biotech.,14:309-314, Sheets et al., 1998, Proc. Nat'l. Acad. Sci., 95:6157-6162,Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381, and Marks et al.,1991, J. Mol. Biol., 222:581). Techniques for the generation and use ofantibody phage libraries are also described in U.S. Pat. Nos. 5,969,108,6,172,197, 5,885,793, 6,521,404; 6,544,731; 6,555,313: 6,582,915;6,593,081; 6,300,064; 6,653,068; 6,706,484; and 7,264,963; and Rothe etal., 2007, J. Mol. Bio., doi:10.1016/j.jmb.2007.12.018 (each of which isincorporated by reference in its entirety). Affinity maturationstrategies and chain shuffling strategies (Marks et al., 1992,Bio/Technology 10:779-783, incorporated by reference in its entirety)are known in the art and may be employed to generate high affinity humanantibodies.

Humanized antibodies can also be made in transgenic mice containinghuman immunoglobulin loci that are capable upon immunization ofproducing the full repertoire of human antibodies in the absence ofendogenous immunoglobulin production. This approach is described in U.S.Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and5,661,016.

This invention also encompasses bispecific antibodies that specificallyrecognize a human frizzled receptor. Bispecific antibodies areantibodies that are capable of specifically recognizing and binding atleast two different epitopes. The different epitopes can either bewithin the same molecule (e.g. the same human frizzled receptor) or ondifferent molecules such that both, for example, the antibodies canspecifically recognize and bind a human frizzled receptor as well as,for example, 1) an effector molecule on a leukocyte such as a T-cellreceptor (e.g. CD3) or Fc receptor (e.g. CD64, CD32, or CD16) or 2) acytotoxic agent as described in detail below. In certain embodiments,the bispecific antibody specifically binds at least one human frizzledreceptor, as well as either VEGF, a Notch ligand, such as a delta-likeligand (for example, DLL4) or jagged, or at least one Notch receptorselected from the group consisting of Notch 1, Notch 2, Notch 3, andNotch 4. Bispecific antibodies can be intact antibodies or antibodyfragments.

Exemplary bispecific antibodies can bind to two different epitopes, atleast one of which originates in a polypeptide of the invention.Alternatively, an anti-antigenic arm of an immunoglobulin molecule canbe combined with an arm which binds to a triggering molecule on aleukocyte such as a T cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG so as to focus cellular defense mechanismsto the cell expressing the particular antigen. Bispecific antibodies canalso be used to direct cytotoxic agents to cells which express aparticular antigen. These antibodies possess an antigen-binding arm andan arm which binds a cytotoxic agent or a radionuclide chelator, such asEOTUBE, DPTA, DOTA, or TETA. Techniques for making bispecific antibodiesare common in the art (Millstein et al., 1983, Nature 305:537-539;Brennan et al., 1985, Science 229:81; Suresh et al, 1986. Methods inEnzymol. 121:120; Traunecker et al., 1991, EMBO J. 10:3655-3659; Shalabyet al., 1992, J. Exp. Med. 175:217-225; Kostelny et al., 1992, J.Immunol. 148:1547-1553; Gruber et al., 1994, J. Immunol. 152:5368; andU.S. Pat. No. 5,731,168). Antibodies with more than two valencies arealso contemplated. For example, trispecific antibodies can be prepared(Tutt et al., J. Immunol. 147:60 (1991)). Thus, in certain embodimentsthe antibodies to human frizzled receptor(s) are multispecific.

Alternatively, in certain alternative embodiments, the FZD-bindingagents of the invention are not bispecific antibodies. In certainembodiments, each of the antigen-binding sites of the antibody isidentical to the other(s).

In certain embodiments, an individual antigen-binding site binds to aregion that is homologous between two or more frizzled receptors (e.g.,FZD1, FZD2, FZD5, FZD7, and/or FZD8). In certain embodiments, anantigen-binding site binds to a region in the BBS that is homologousbetween two or more frizzled receptors.

In certain embodiments, the antibodies (or other polypeptides) describedherein may be monospecific. For example, in certain embodiments, each ofthe one or more antigen-binding sites that an antibody contains iscapable of binding (or binds) the same one or more human FZD receptors(e.g., FZD1, FZD2, FZD5, FZD7, or FZD8, or a homologous epitope on somecombination of the FZDs). In certain embodiments, an antigen-bindingsite of a monospecific antibody described herein is capable of binding(or binds) one, two, three, four, or five (or more) human frizzledreceptors.

In certain embodiments are provided an antibody fragment to, forexample, increase tumor penetration. Various techniques are known forthe production of antibody fragments. Traditionally, these fragments arederived via proteolytic digestion of intact antibodies (for exampleMorimoto et al., 1993, Journal of Biochemical and Biophysical Methods24:107-117; Brennan et al., 1985, Science, 229:81). In certainembodiments, antibody fragments are produced recombinantly. Fab, Fv, andscFv antibody fragments can all be expressed in and secreted from E.coli or other host cells, thus allowing the production of large amountsof these fragments. Such antibody fragments can also be isolated fromthe antibody phage libraries discussed above. The antibody fragment canalso be linear antibodies as described in U.S. Pat. No. 5,641,870, forexample, and can be monospecific or bispecific. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner.

According to the present invention, techniques can be adapted for theproduction of single-chain antibodies specific to one or more humanfrizzled receptors (see U.S. Pat. No. 4,946,778). In addition, methodscan be adapted for the construction of Fab expression libraries (Huse,et al., Science 246:1275-1281 (1989)) to allow rapid and effectiveidentification of monoclonal Fab fragments with the desired specificityfor a FZD receptor, or derivatives, fragments, analogs or homologsthereof. Antibody fragments may be produced by techniques in the artincluding, but not limited to: (a) a F(ab′)2 fragment produced by pepsindigestion of an antibody molecule; (b) a Fab fragment generated byreducing the disulfide bridges of an F(ab′)2 fragment, (c) a Fabfragment generated by the treatment of the antibody molecule with papainand a reducing agent, and (d) Fv fragments.

It can further be desirable, especially in the case of antibodyfragments, to modify an antibody in order to increase its serumhalf-life. This can be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment by mutationof the appropriate region in the antibody fragment or by incorporatingthe epitope into a peptide tag that is then fused to the antibodyfragment at either end or in the middle (e.g., by DNA or peptidesynthesis).

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune cells to unwanted cells (U.S. Pat. No. 4,676,980). It iscontemplated that the antibodies can be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins can be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

For the purposes of the present invention, it should be appreciated thatmodified antibodies can comprise any type of variable region thatprovides for the association of the antibody with the polypeptides of ahuman FZD receptor. In this regard, the variable region may comprise orbe derived from any type of mammal that can be induced to mount ahumoral response and generate immunoglobulins against the desired tumorassociated antigen. As such, the variable region of the modifiedantibodies can be, for example, of human, murine, non-human primate(e.g. cynomolgus monkeys, macaques, etc.) or lupine origin. In someembodiments both the variable and constant regions of the modifiedimmunoglobulins are human. In other embodiments the variable regions ofcompatible antibodies (usually derived from a non-human source) can beengineered or specifically tailored to improve the binding properties orreduce the immunogenicity of the molecule. In this respect, variableregions useful in the present invention can be humanized or otherwisealtered through the inclusion of imported amino acid sequences.

In certain embodiments, the variable domains in both the heavy and lightchains are altered by at least partial replacement of one or more CDRsand, if necessary, by partial framework region replacement and sequencechanging. Although the CDRs may be derived from an antibody of the sameclass or even subclass as the antibody from which the framework regionsare derived, it is envisaged that the CDRs will be derived from anantibody of different class and preferably from an antibody from adifferent species. It may not be necessary to replace all of the CDRswith the complete CDRs from the donor variable region to transfer theantigen binding capacity of one variable domain to another. Rather, itmay only be necessary to transfer those residues that are necessary tomaintain the activity of the antigen binding site. Given theexplanations set forth in U.S. Pat. Nos. 5,585,089, 5,693,761 and5,693,762, it will be well within the competence of those skilled in theart, either by carrying out routine experimentation or by trial anderror testing to obtain a functional antibody with reducedimmunogenicity.

Alterations to the variable region notwithstanding, those skilled in theart will appreciate that the modified antibodies of this invention willcomprise antibodies (e.g. full-length antibodies or immunoreactivefragments thereof) in which at least a fraction of one or more of theconstant region domains has been deleted or otherwise altered so as toprovide desired biochemical characteristics such as increased tumorlocalization or reduced serum half-life when compared with an antibodyof approximately the same immunogenicity comprising a native orunaltered constant region. In some embodiments, the constant region ofthe modified antibodies will comprise a human constant region.Modifications to the constant region compatible with this inventioncomprise additions, deletions or substitutions of one or more aminoacids in one or more domains. That is, the modified antibodies disclosedherein may comprise alterations or modifications to one or more of thethree heavy chain constant domains (CH1, CH2 or CH3) and/or to the lightchain constant domain (CL). In some embodiments, modified constantregions wherein one or more domains are partially or entirely deletedare contemplated. In some embodiments, the modified antibodies willcomprise domain deleted constructs or variants wherein the entire CH2domain has been removed (ΔCH2 constructs). In some embodiments, theomitted constant region domain will be replaced by a short amino acidspacer (e.g. 10 residues) that provides some of the molecularflexibility typically imparted by the absent constant region.

Besides their configuration, it is known in the art that the constantregion mediates several effector functions. For example, binding of theCl component of complement to antibodies activates the complementsystem. Activation of complement is important in the opsonisation andlysis of cell pathogens. The activation of complement also stimulatesthe inflammatory response and can also be involved in autoimmunehypersensitivity. Further, antibodies bind to cells via the Fc region,with a Fc receptor site on the antibody Fc region binding to a Fereceptor (FcR) on a cell. There are a number of Fc receptors which arespecific for different classes of antibody, including IgG (gammareceptors), IgE (eta receptors), IgA (alpha receptors) and IgM (mureceptors). Binding of antibody to Fc receptors on cell surfacestriggers a number of important and diverse biological responsesincluding engulfment and destruction of antibody-coated particles,clearance of immune complexes, lysis of antibody-coated target cells bykiller cells (called antibody-dependent cell-mediated cytotoxicity, orADCC), release of inflammatory mediators, placental transfer and controlof immunoglobulin production.

In certain embodiments, the FZD-binding antibodies provide for alteredeffector functions that, in turn, affect the biological profile of theadministered antibody. For example, the deletion or inactivation(through point mutations or other means) of a constant region domain mayreduce Fc receptor binding of the circulating modified antibody therebyincreasing tumor localization. In other cases it may be that constantregion modifications, consistent with this invention, moderatecomplement binding and thus reduce the serum half life and nonspecificassociation of a conjugated cytotoxin. Yet other modifications of theconstant region may be used to eliminate disulfide linkages oroligosaccharide moieties that allow for enhanced localization due toincreased antigen specificity or antibody flexibility. Similarly,modifications to the constant region in accordance with this inventionmay easily be made using well known biochemical or molecular engineeringtechniques well within the purview of the skilled artisan.

In certain embodiments, a FZD-binding agent that is an antibody does nothave one or more effector functions. For instance, in some embodiments,the antibody has no antibody-dependent cellular cytoxicity (ADCC)activity and/or no complement-dependent cytoxicity (CDC) activity. Incertain embodiments, the antibody does not bind to an Fc receptor and/orcomplement factors. In certain embodiments, the antibody has no effectorfunction.

It will be noted that in certain embodiments, the modified antibodiesmay be engineered to fuse the CH3 domain directly to the hinge region ofthe respective modified antibodies. In other constructs it may bedesirable to provide a peptide spacer between the hinge region and themodified CH2 and/or CH3 domains. For example, compatible constructscould be expressed wherein the CH2 domain has been deleted and theremaining CH3 domain (modified or unmodified) is joined to the hingeregion with a 5-20 amino acid spacer. Such a spacer may be added, forinstance, to ensure that the regulatory elements of the constant domainremain free and accessible or that the hinge region remains flexible.However, it should be noted that amino acid spacers can, in some cases,prove to be immunogenic and elicit an unwanted immune response againstthe construct. Accordingly, in certain embodiments, any spacer added tothe construct will be relatively non-immunogenic, or even omittedaltogether, so as to maintain the desired biochemical qualities of themodified antibodies.

Besides the deletion of whole constant region domains, it will beappreciated that the antibodies of the present invention may be providedby the partial deletion or substitution of a few or even a single aminoacid. For example, the mutation of a single amino acid in selected areasof the CH2 domain may be enough to substantially reduce Fc binding andthereby increase tumor localization. Similarly, it may be desirable tosimply delete that part of one or more constant region domains thatcontrol the effector function (e.g. complement CLQ binding) to bemodulated. Such partial deletions of the constant regions may improveselected characteristics of the antibody (serum half-life) while leavingother desirable functions associated with the subject constant regiondomain intact. Moreover, as alluded to above, the constant regions ofthe disclosed antibodies may be modified through the mutation orsubstitution of one or more amino acids that enhances the profile of theresulting construct. In this respect it may be possible to disrupt theactivity provided by a conserved binding site (e.g. Fc binding) whilesubstantially maintaining the configuration and immunogenic profile ofthe modified antibody. Certain embodiments can comprise the addition ofone or more amino acids to the constant region to enhance desirablecharacteristics such as decreasing or increasing effector function orprovide for more cytotoxin or carbohydrate attachment. In suchembodiments it can be desirable to insert or replicate specificsequences derived from selected constant region domains.

The present invention further embraces variants and equivalents whichare substantially homologous to the chimeric, humanized and humanantibodies, or antibody fragments thereof, set forth herein. These cancontain, for example, conservative substitution mutations, i.e. thesubstitution of one or more amino acids by similar amino acids. Forexample, conservative substitution refers to the substitution of anamino acid with another within the same general class such as, forexample, one acidic amino acid with another acidic amino acid, one basicamino acid with another basic amino acid or one neutral amino acid byanother neutral amino acid. What is intended by a conservative aminoacid substitution is well known in the art.

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent. Cytotoxic agents includechemotherapeutic agents, growth inhibitory agents, toxins (e.g., anenzymatically active toxin of bacterial, fungal, plant, or animalorigin, or fragments thereof), radioactive isotopes (i.e., aradioconjugate), etc. Chemotherapeutic agents useful in the generationof such immunoconjugates include, for example, methotrexate, adriamicin,doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or otherintercalating agents. Enzymatically active toxins and fragments thereofthat can be used include diphtheria A chain, nonbinding active fragmentsof diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain,modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthinproteins. Phytolaca americana proteins (PAPI, PAPII, and PAP-S),momordica charantia inhibitor, curcin, crotin, sapaonaria officinalisinhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, andthe tricothecenes. A variety of radionuclides are available for theproduction of radioconjugated antibodies including 212Bi. 131I. 131In,90Y, and 186Re. Conjugates of the antibody and cytotoxic agent are madeusing a variety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such asbis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Conjugates of an antibody and one ormore small molecule toxins, such as a calicheamicin, maytansinoids, atrichothene, and CC1065, and the derivatives of these toxins that havetoxin activity, can also be used.

Conjugate antibodies are composed of two covalently joined antibodies.Such antibodies have, for example, been proposed to target immune cellsto unwanted cells (U.S. Pat. No. 4,676,980). It is contemplated that theantibodies can be prepared in vitro using known methods in syntheticprotein chemistry, including those involving crosslinking agents. Forexample, immunotoxins can be constructed using a disulfide exchangereaction or by forming a thioether bond. Examples of suitable reagentsfor this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

Regardless of how useful quantities are obtained, the antibodies of thepresent invention can be used in any one of a number of conjugated (i.e.an immunoconjugate) or unconjugated forms. Alternatively, the antibodiesof this invention can be used in a nonconjugated or “naked” form. Incertain embodiments, the antibodies are used in nonconjugated form toharness the subject's natural defense mechanisms includingcomplement-dependent cytotoxicity (CDC) and antibody dependent cellulartoxicity (ADCC) to eliminate the malignant cells. In some embodiments,the antibodies can be conjugated to radioisotopes, such as ⁹⁰Y, ¹²⁵I,¹³¹I, ¹²³I, ¹¹¹In, ¹⁰⁵Rh, ¹⁵³Sm, ⁶⁷Cu, ⁶⁷Ga, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re and¹⁸⁸Re using anyone of a number of well known chelators or directlabeling. In other embodiments, the disclosed compositions can compriseantibodies coupled to drugs, prodrugs or biological response modifierssuch as methotrexate, adriamycin, and lymphokines such as interferon.Still other embodiments of the present invention comprise the use ofantibodies conjugated to specific biotoxins such as ricin or diptheriatoxin. In yet other embodiments the modified antibodies can be complexedwith other immunologically active ligands (e.g. antibodies or fragmentsthereof) wherein the resulting molecule binds to both the neoplasticcell and an effector cell such as a T cell. The selection of whichconjugated or unconjugated modified antibody to use will depend of thetype and stage of cancer, use of adjunct treatment (e.g., chemotherapyor external radiation) and patient condition. It will be appreciatedthat one skilled in the art could readily make such a selection in viewof the teachings herein.

The polypeptides of the present invention can be recombinantpolypeptides, natural polypeptides, or synthetic polypeptides comprisingan antibody, or fragment thereof, against a human FZD receptor. It willbe recognized in the art that some amino acid sequences of the inventioncan be varied without significant effect of the structure or function ofthe protein. Thus, the invention further includes variations of thepolypeptides which show substantial activity or which include regions ofan antibody, or fragment thereof, against a human FZD receptor protein.Such mutants include deletions, insertions, inversions, repeats, andtype substitutions.

The polypeptides and analogs can be further modified to containadditional chemical moieties not normally part of the protein. Thosederivatized moieties can improve the solubility, the biological halflife or absorption of the protein. The moieties can also reduce oreliminate any desirable side effects of the proteins and the like. Anoverview for those moieties can be found in REMINGTON'S PHARMACEUTICALSCIENCES, 20th ed., Mack Publishing Co., Easton, Pa. (2000).

The isolated polypeptides described herein can be produced by anysuitable method known in the art. Such methods range from direct proteinsynthetic methods to constructing a DNA sequence encoding isolatedpolypeptide sequences and expressing those sequences in a suitabletransformed host. In some embodiments, a DNA sequence is constructedusing recombinant technology by isolating or synthesizing a DNA sequenceencoding a wild-type protein of interest. Optionally, the sequence canbe mutagenized by site-specific mutagenesis to provide functionalanalogs thereof. See, e.g. Zoeller et al., Proc. Nat'l. Acad. Sci. USA81:5662-5066 (1984) and U.S. Pat. No. 4,588,585.

In some embodiments a DNA sequence encoding a polypeptide of interestwould be constructed by chemical synthesis using an oligonucleotidesynthesizer. Such oligonucleotides can be designed based on the aminoacid sequence of the desired polypeptide and selecting those codons thatare favored in the host cell in which the recombinant polypeptide ofinterest will be produced. Standard methods can be applied to synthesizean isolated polynucleotide sequence encoding an isolated polypeptide ofinterest. For example, a complete amino acid sequence can be used toconstruct a back-translated gene. Further, a DNA oligomer containing anucleotide sequence coding for the particular isolated polypeptide canbe synthesized. For example, several small oligonucleotides coding forportions of the desired polypeptide can be synthesized and then ligated.The individual oligonucleotides typically contain 5′ or 3′ overhangs forcomplementary assembly.

Once assembled (by synthesis, site-directed mutagenesis or anothermethod), the polynucleotide sequences encoding a particular isolatedpolypeptide of interest will be inserted into an expression vector andoperatively linked to an expression control sequence appropriate forexpression of the protein in a desired host. Proper assembly can beconfirmed by nucleotide sequencing, restriction mapping, and expressionof a biologically active polypeptide in a suitable host. As is wellknown in the art, in order to obtain high expression levels of atransfected gene in a host, the gene must be operatively linked totranscriptional and translational expression control sequences that arefunctional in the chosen expression host.

In certain embodiments, recombinant expression vectors are used toamplify and express DNA encoding antibodies, or fragments thereof,against human frizzled receptors. Recombinant expression vectors arereplicable DNA constructs which have synthetic or cDNA-derived DNAfragments encoding a polypeptide chain of an anti-FZD antibody, orfragment thereof, operatively linked to suitable transcriptional ortranslational regulatory elements derived from mammalian, microbial,viral or insect genes. A transcriptional unit generally comprises anassembly of (1) a genetic element or elements having a regulatory rolein gene expression, for example, transcriptional promoters or enhancers,(2) a structural or coding sequence which is transcribed into mRNA andtranslated into protein, and (3) appropriate transcription andtranslation initiation and termination sequences, as described in detailbelow. Such regulatory elements can include an operator sequence tocontrol transcription. The ability to replicate in a host, usuallyconferred by an origin of replication, and a selection gene tofacilitate recognition of transformants can additionally beincorporated. DNA regions are operatively linked when they arefunctionally related to each other. For example, DNA for a signalpeptide (secretory leader) is operatively linked to DNA for apolypeptide if it is expressed as a precursor which participates in thesecretion of the polypeptide; a promoter is operatively linked to acoding sequence if it controls the transcription of the sequence; or aribosome binding site is operatively linked to a coding sequence if itis positioned so as to permit translation. Structural elements intendedfor use in yeast expression systems include a leader sequence enablingextracellular secretion of translated protein by a host cell.Alternatively, where recombinant protein is expressed without a leaderor transport sequence, it can include an N-terminal methionine residue.This residue can optionally be subsequently cleaved from the expressedrecombinant protein to provide a final product.

The choice of expression control sequence and expression vector willdepend upon the choice of host. A wide variety of expression host/vectorcombinations can be employed. Useful expression vectors for eukaryotichosts, include, for example, vectors comprising expression controlsequences from SV40, bovine papilloma virus, adenovims andcytomegalovirus. Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from Esherichia coli,including pCR 1, pBR322, pMB9 and their derivatives, wider host rangeplasmids, such as M13 and filamentous single-stranded DNA phages.

Suitable host cells for expression of a FZD-binding polypeptide orantibody (or a FZD protein to use as an antigen) include prokaryotes,yeast, insect or higher eukaryotic cells under the control ofappropriate promoters. Prokaryotes include gram negative or grampositive organisms, for example E. coli or bacilli. Higher cukaryoticcells include established cell lines of mammalian origin as describedbelow. Cell-free translation systems could also be employed. Appropriatecloning and expression vectors for use with bacterial, fungal, yeast,and mammalian cellular hosts are described by Pouwels et al. (CloningVectors: A Laboratory Manual, Elsevier, N.Y., 1985), the relevantdisclosure of which is hereby incorporated by reference. Additionalinformation regarding methods of protein production, including antibodyproduction, can be found, e.g., in U.S. Patent Publication No.2008/0187954, U.S. Pat. Nos. 6,413,746 and 6,660,501, and InternationalPatent Publication No. WO 04009823, each of which is hereby incorporatedby reference herein in its entirety.

Various mammalian or insect cell culture systems are also advantageouslyemployed to express recombinant protein. Expression of recombinantproteins in mammalian cells can be performed because such proteins aregenerally correctly folded, appropriately modified and completelyfunctional. Examples of suitable mammalian host cell lines include theCOS-7 lines of monkey kidney cells, described by Gluzman (Cell 23:175,1981), and other cell lines capable of expressing an appropriate vectorincluding, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO).HeLa and BHK cell lines. Mammalian expression vectors can comprisenontranscribed elements such as an origin of replication, a suitablepromoter and enhancer linked to the gene to be expressed, and other 5′or 3′ flanking nontranscribed sequences, and 5′ or 3′ nontranslatedsequences, such as necessary ribosome binding sites, a polyadenylationsite, splice donor and acceptor sites, and transcriptional terminationsequences. Baculovirus systems for production of heterologous proteinsin insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47(1988).

The proteins produced by a transformed host can be purified according toany suitable method. Such standard methods include chromatography (e.g.,ion exchange, affinity and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for protein purification. Affinity tags such as hexahistidine,maltose binding domain, influenza coat sequence andglutathione-S-transferase can be attached to the protein to allow easypurification by passage over an appropriate affinity column. Isolatedproteins can also be physically characterized using such techniques asproteolysis, nuclear magnetic resonance and x-ray crystallography.

For example, supernatants from systems which secrete recombinant proteininto culture media can be first concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon ultrafiltration unit. Following the concentrationstep, the concentrate can be applied to a suitable purification matrix.Alternatively, an anion exchange resin can be employed, for example, amatrix or substrate having pendant diethylaminoethyl (DEAE) groups. Thematrices can be acrylamide, agarose, dextran, cellulose or other typescommonly employed in protein purification. Alternatively, a cationexchange step can be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a FZD-binding agent. Some or all of theforegoing purification steps, in various combinations, can also beemployed to provide a homogeneous recombinant protein.

Recombinant protein produced in bacterial culture can be isolated, forexample, by initial extraction from cell pellets, followed by one ormore concentration, salting-out, aqueous ion exchange or size exclusionchromatography steps. High performance liquid chromatography (HPLC) canbe employed for final purification steps. Microbial cells employed inexpression of a recombinant protein can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

Methods known in the art for purifying antibodies and other proteinsalso include, for example, those described in U.S. Patent PublicationNo. 2008/0312425, 2008/0177048, and 2009/0187005, each of which ishereby incorporated by reference herein in its entirety.

In certain embodiments, the FZD-binding agent is a polypeptide that isnot an antibody. A variety of methods for identifying and producingnon-antibody polypeptides that bind with high affinity to a proteintarget are known in the art. See, e g., Skerra, Curr. Opin. Biotechnol.,18:295-304 (2007), Hosse et al., Protein Science. 15:14-27 (2006), Gillet al. Curr. Opin. Biotechnol., 17:653-658 (2006), Nygren, FEBS J.,275:2668-76 (2008), and Skerra, FEBS J., 275:2677-83 (2008), each ofwhich is incorporated by reference herein in its entirety

In certain embodiments, phage display technology has been used toidentify/produce the FZD-binding polypeptide. In certain embodiments,the polypeptide comprises a protein scaffold of a type selected from thegroup consisting of protein A, a lipocalin, a fribronectin domain, anankyrin consensus repeat domain, and thioredoxin.

In certain embodiments, the polypeptide has been naturally orunnaturally modified. By way of non-limiting example, the polypeptidemay be labeled. In certain embodiments, the polypeptide is glycosylated,pegylated, phosphorylated, or acetylated, amidated. In certainembodiments, the modifications increase stability and/or the in vivohalf-life of the polypeptide. In certain embodiments, the polypeptidesare cyclic. In certain further embodiments, the polypeptides compriseone or more N-methyl amino acids.

In certain embodiments, the FZD-binding agent is (or comprises) apolypeptide comprising (a) a sequence selected from the group consistingof SEQ ID NOs:98-112 or (b) a sequence having at least about 80% aminoacid sequence identity to a sequence selected from the group consistingof SEQ ID NOs:98-111. In certain embodiments, the polypeptide comprisesa sequence having at least about 85%, at least about 88%, or at leastabout 90% amino acid sequence identity to a sequence selected from thegroup consisting of SEQ ID NOs:98-11. In certain embodiments, thepolypeptides comprise, consist essentially of, or consist of a cyclicpeptide selected from the group consisting of SEQ ID NOs:98-112. Incertain embodiments, the sequence is SEQ ID NO:108. In certainalternative embodiments, the sequence is SEQ ID NO: 112.

In certain alternative embodiments, the FZD-binding agent is (orcomprises) a polypeptide having at least about 80%, at least about 85%,at least about 88%, or at least about 90% amino acid sequence identityto a sequence selected from the group consisting of SEQ ID NOs:113-116.In certain alternative embodiments, the FZD-binding agent is (orcomprises) a polypeptide comprising, consisting essentially of, orconsisting of a sequence selected from the group consisting of SEQ IDNOs: 113-116.

In certain embodiments, the FZD-binding polypeptide may be less thanabout 500 amino acids in length, less than about 200 amino acids inlength, less than about 100 amino acids in length, less than about 50amino acids in length, less than about 20 amino acids amino acids inlength, or less than about 15 amino acids in length. Such polypeptidesmay be at least about 3, at least about 5, or at least about 7 aminoacids in length. Accordingly, in certain embodiments the polypeptide maybe between about 5 and about 20 amino acids in length. In someembodiments, the polypeptide is between about 7 and about 15 amino acidsin length.

In some embodiments, the agent is a non-protein molecule. In certainembodiments, the agent is a small molecule. Combinatorial chemistrylibraries and techniques useful in the identification of non-proteinFZD-binding agents are known to those skilled in the art. See, e.g.,Kennedy et al., J. Comb. Chem., 10:345-354 (2008), Dolle et al, J. Comb.Chem., 9:855-902 (2007), and Bhattacharyya. Curr. Med. Chem., 8:1383-404(2001), each of which is incorporated by reference herein in itsentirety. In certain further embodiments, the agent is a carbohydrate, aglycosaminoglycan, a glycoprotein, or a proteoglycan.

In certain embodiments, the agent comprises a lipid. For instance, insome instances, the agent may comprise a fatty acid or a derivativethereof. In certain embodiments, the fatty acid is myristoleic acid,palmitoleic acid, sapienic acid, oleic acid, linoleic acid, α-linolenicacid, arachidonic acid, eicosapentaenoic acid, erucic acid, ordocosahexaenoic acid. Because the cleft of the BBS is hydrophobic,agents comprising one or more lipids are particularly suitable fortargeting the cleft.

In certain embodiments, the agent is a nucleic acid aptamer. Aptamersare polynucleotide molecules that have been selected (e.g., from randomor mutagenized pools) on the basis of their ability to bind to anothermolecule. In some embodiments, the aptamer comprises a DNApolynucleotide. In certain alternative embodiments, the aptamercomprises an RNA polynucleotide. In certain embodiments, the aptamercomprises one or more modified nucleic acid residues. Methods ofgenerating and screening nucleic acid aptamers for binding to proteinsare well known in the art. See, e.g., U.S. Pat. No. 5,270,163, U.S. Pat.No. 5,683,867, U.S. Pat. No. 5,763,595, U.S. Pat. No. 6,344,321, U.S.Pat. No. 7,368,236, U.S. Pat. No. 5,582,981, U.S. Pat. No. 5,756,291,U.S. Pat. No. 5,840,867. U.S. Pat. No. 7,312,325, U.S. Pat. No.7,329,742. International Patent Publication No. WO 02/077262,International Patent Publication No. WO 03/070984, U.S. PatentApplication Publication No. 2005/0239134, U.S. Patent ApplicationPublication No. 2005/0124565, and U.S. Patent Application PublicationNo. 2008/0227735, each of which is incorporated by reference herein inits entirety.

In certain embodiments, the agent has a molecular weight of at leastabout 100 daltons. In certain embodiments, the agent has a molecularweight of at least about 1000 daltons. In certain embodiments, the agenthas a molecular weight of less than about 2000 daltons or less thanabout 1000 daltons.

The present invention further provides methods of screening agents forefficacy in inhibiting Wnt signaling, for anti-tumor efficacy, and/orefficacy against cancer stem cells. These methods include, but are notlimited to, methods comprising comparing the levels of one or moredifferentiation markers in a first solid tumor that has been exposed tothe agent relative to the levels of the one or more differentiationmarkers in a second solid tumor that has not been exposed to the agent.In certain embodiments, these methods include (a) exposing a first solidtumor, but not a second solid tumor, to the agent: (b) assessing thelevels of one or more differentiation markers in the first and secondsolid tumors; and (c) comparing the levels of the one or moredifferentiation markers in the first and second solid tumors. In certainembodiments, the agent is an inhibitor of the canonical Wnt signalingpathway, and Lor inhibits binding of one or more human Wnt proteins toone or more human frizzled receptors. In certain embodiments, the agentis an antibody that specifically binds to one or more human frizzledreceptor. In certain embodiments, increased levels of one or moredifferentiation markers in the first solid tumor relative to the secondsolid tumor indicates efficacy against solid tumor stem cells. Incertain alternative embodiments, decreased levels of one or moredifferentiation markers (i.e., negative markers for differentiation) inthe first solid tumor relative to the second solid tumor indicatesefficacy against solid tumor stem cells. In certain embodiments, thesolid tumor is a pancreatic tumor. In certain embodiments, the solidtumor is a pancreatic tumor and the one or more differentiation markersmay comprise one or more mucins (e.g., Muc16) and/or chromogranin A(CHGA). In certain alternative embodiments, the solid tumor is a colontumor. In some embodiments, the solid tumor is a colon tumor and the oneor more differentiation markers comprise cytokeratin 7. Other potentialdifferentiation markers for pancreas and colon as well as other tumortypes are known to those skilled in the art. The usefulness of potentialdifferentiation markers in a screening method can be readily assessed byone skilled in the art by treating the desired tumor type with one ormore of the anti-FZD antibodies disclosed herein such as 18R5 and/or44R24 and then assessing for changes in expression of the marker by thetreated tumor relative to control. Non-limiting examples of suchmethods, can for instance, be found in the specific Examples below.

Useful reagents for additional screening assays include mutant FZDderivatives. For instance, polypeptides comprising the Fri domain of afrizzled receptor in which one or more (e.g., 1, 2, 3, 4, 5, 6, 7 ormore) amino acid substitutions have been made in the Biological BindingSite (BBS). In certain embodiments, the mutant polypeptides exhibit areduced binding affinity to a Wnt (e.g., Wnt3a), relative to the bindingaffinity that a polypeptide comprising the wild-type Fri domain of thefrizzled receptor has for the Wnt. In certain embodiments, the wild-typeFri domain comprises a minimum Fri domain sequence such as a sequenceselected from the group consisting of SEQ ID NOs. 117-126.Alternatively, the wild-type Fri domain comprises a polypeptide having aFZD Fri domain sequence selected from the group consisting of SEQ IDNOs. 28, 32, 36, 40, 44, 48, 52, 56, 60, and 64. In certain embodiments,one or more of the amino acid substitutions can be made at the site of acleft residue selected from Table 16 and/or Table 17. In certainembodiments, the Fri domain is a FZD4, FZD5, or FZD8 Fri domain. Incertain embodiments, the Fri domain is a FZD8 Fri domain comprising oneor more substitutions at F72, L75, 178, or L121. In certain embodiments,the FZD8 Fri domain comprising a mutation at L75. An amino acidsubstitution(s) in the cleft of the BBS may, for example, be an aminoacid residue(s) that is less hydrophobic than the amino acid residue atthat same site in the wild-type FZD sequence. Alternatively, the one ormore substitutions may be made in the top overhang and/or the bottomoverhang of the BBS. In certain embodiments, the mutant FZD polypeptideis soluble. In certain embodiments, the mutant FZD polypeptide is asoluble FZD receptor and may comprise an Fc region, such as a human Fcregion.

In certain embodiments, the invention provides a polypeptide comprisingthe Fri domain of FZD8 in which one or more substitutions have been madein the BBS which impair binding of FZD8 to Wnt. For example, the one ormore substitutions may have been made in the cleft of the BBS. Incertain embodiments, one or more substitutions have been made at F72,L75, I78, and/or L121. Non-limiting examples of possible substitutionsinclude F72Y, L75S, 178S, or L121S. In certain embodiments, asubstitution has been made at L75 (e.g., L75S).Assays for screening foragents which bind to the BBS of a FZD receptor (e.g., a human FZDreceptor), inhibit Wnt signaling and/or can be used as therapeuticagents, such as anti-cancer agents, include assays in which a compoundis screened for preferential binding to a wild-type FZD Fri domainsequence relative to a mutant FZD polypeptide in which one or more aminoacid substitutions have been made in the BBS. For example, the inventionprovides a method of screening a compound for possible activity as aninhibitor of Wnt signaling. In one embodiment, the method comprisesdetermining the binding affinity (qualitatively or quantitatively) of acompound for a first polypeptide comprising the Fri domain of a frizzledreceptor, wherein the Fri domain comprises one or more substitutions inthe Biological Binding Site (BBS). The method further comprisesdetermining the binding affinity of the compound for a secondpolypeptide comprising the wild-type sequence of the Fri domain of thehuman Frizzled receptor of the first polypeptide, and then comparing thebinding affinity of the compound for the first polypeptide with thebinding affinity of the compound for the second polypeptide. If thecompound has greater affinity for the second (wild-type FZD Fri domain)polypeptide than the first (mutant BBS) polypeptide, the compound isidentified as a possible inhibitor of Wnt signaling (and candidateanti-cancer agent).

In certain embodiments, the FZD receptors in the above-described assaysor other assays described herein are human FZD receptors. A human FZDreceptor may be FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7. FZD8, FZD9, orFZD10. Likewise, in certain embodiments of the aforementioned assays, orother assays described elsewhere herein, the Wnt protein is a human Wntprotein. For example, in certain embodiments, the Wnt protein is humanWnt3a, or another human Wnt protein described elsewhere herein.

The invention further provides additional polypeptides (e.g.,FZD-binding polypeptides) useful in screening assays. For instance, theinvention provides polypeptides comprising a sequence selected from thegroup consisting of SEQ ID NOs:98-112. In certain embodiments, thepolypeptides comprise, consist essentially of, or consist of a cyclicpeptide selected from the group consisting of SEQ ID NOs:98-112. Theinvention also provides isolated polypeptides comprising, consistingessentially of, or consisting of a sequence selected from the groupconsisting of SEQ ID NOs:113-116. In certain embodiments, the sequenceis SEQ ID NO:108. In certain embodiments, the sequence is SEQ ID NO:112. In certain alternative embodiments, the sequence is SEQ ID NO:113.The polypeptides used in the competitive binding assays and/ordisplacement assays are, in some instances, labeled or modified.Generally, any of the FZD-binding agents described herein can be used insuch assays. In certain alternative embodiments, the FZD-bindingpolypeptide used in a competitive binding assay is the antibody 18R5.

Screening methods involving the use of the FZD-binding polypeptidesdescribed in the preceding paragraph or elsewhere herein are alsoprovided (e.g. FZD-binding agents that bind to one or more residues inthe cleft of the BBS). In one embodiment, a method of screening acompound for possible activity as an inhibitor of Wnt signalingcomprises determining whether the compound can compete with aFZD-binding polypeptide for binding to a human FZD receptor (and/ordisplace the polypeptide from the FZD receptor to which it is bound.).In such a method, the ability of the compound to compete for bindingwith the polypeptide (and/or displace the polypeptide from the FZDreceptor to which it is bound) indicates the compound has possibleactivity as an inhibitor of Wnt signaling. In certain embodiments, theFZD receptor is FZD8. In certain embodiments, the human FZD receptormolecule actually used in the screening assay is in a soluble form(e.g., a FZD-Fc molecule such as FZD8Fc, which comprises theextracellular domain or at least the Fri domain of the FZD protein fusedto an Fc region).

In certain embodiments, the screening assays and/or other assaysdescribed herein have been used to identify the FZD-binding agents. Forexample, the agent may have been identified by a competitive binding ordisplacement assay using a polypeptide comprising one of SEQ IDNOs:98-116. Alternatively, the agent may have, for example beenidentified by reduced binding to a FZD containing one or moresubstitutions in the BBS relative to the wild-type FZD.

III. POLYNUCLEOTIDES

In certain embodiments, the invention encompasses polynucleotidescomprising polynucleotides that encode a polypeptide that specificallybinds a human FZD receptor or a fragment of such a polypeptide. Forexample, the invention provides a polynucleotide comprising a nucleicacid sequence that encodes an antibody to a human frizzled receptor orencodes a fragment of such an antibody. The polynucleotides of theinvention can be in the form of RNA or in the form of DNA. DNA includescDNA, genomic DNA, and synthetic DNA; and can be double-stranded orsingle-stranded, and if single stranded can be the coding strand ornon-coding (anti-sense) strand.

In certain embodiments, the polynucleotides are isolated. In certainembodiments, the polynucleotides are substantially pure.

The invention provides a polynucleotide comprising a polynucleotideencoding a polypeptide comprising a sequence selected from the groupconsisting of SEQ ID NOs:10, 12, 14. The invention further provides apolynucleotide comprising a polynucleotide encoding a polypeptidecomprising a sequence selected from the group consisting of SEQ ID NOs:85-86. A polynucleotide comprising polynucleotides encoding apolypeptide comprising SEQ ID NOs: 11, 13, or 15 is also provided.

Also provided is a polynucleotide comprising a polynucleotide thatencodes a polypeptide comprising a sequence selected from the groupconsisting of SEQ ID NOs:98-111 and SEQ ID NOs:113-116. In certainembodiments, the polynucleotide comprises a polynucleotide that encodesSEQ ID NO: 108.

The invention further provides a polynucleotide comprising a sequenceselected from the group consisting of SEQ ID NOs:17, 19, and 21.Alternatively, in certain embodiments, the polynucleotide may comprise asequence selected from the group consisting of SEQ ID NOs: 87-90, 92,and 94-95. Polynucleotide sequences comprising SEQ ID NO: 18, 20, or 22are also provided.

Also provided is a polynucleotide that comprises a polynucleotide thathybridizes to a polynucleotide having the sequence of SEQ ID NO: 17, 19,or 21 and/or to a polynucleotide encoding a polypeptide having thesequence of SEQ ID NO:10, 12, or 14. Also provided is a polynucleotidethat comprises a polynucleotide that hybridizes to a polynucleotidehaving a sequence selected from the group consisting of SEQ ID NOs:87-90, 92, and 94-95 and/or to a polynucleotide encoding a polypeptidehaving the sequence of SEQ ID NO: 85 or 86. In certain embodiments, thehybridization is under conditions of high stringency.

In certain embodiments the polynucleotides comprise the coding sequencefor the mature polypeptide fused in the same reading frame to apolynucleotide which aids, for example, in expression and secretion of apolypeptide from a host cell (e.g. a leader sequence which functions asa secretory sequence for controlling transport of a polypeptide from thecell). The polypeptide having a leader sequence is a preprotein and canhave the leader sequence cleaved by the host cell to form the matureform of the polypeptide. The polynucleotides can also encode for aproprotein which is the mature protein plus additional 5′ amino acidresidues. A mature protein having a prosequence is a proprotein and isan inactive form of the protein. Once the prosequence is cleaved anactive mature protein remains.

In certain embodiments the polynucleotides comprise the coding sequencefor the mature polypeptide fused in the same reading frame to a markersequence that allows, for example, for purification of the encodedpolypeptide. For example, the marker sequence can be a hexa-histidinetag supplied by a pQE-9 vector to provide for purification of the maturepolypeptide fused to the marker in the case of a bacterial host, or themarker sequence can be a hemagglutinin (HA) tag derived from theinfluenza hemagglutinin protein when a mammalian host (e.g. COS-7 cells)is used.

The present invention further relates to variants of the hereinabovedescribed polynucleotides encoding, for example, fragments, analogs, andderivatives.

In certain embodiments, the present invention provides isolatedpolynucleotides comprising polynucleotides having a nucleotide sequenceat least 80% identical, at least 85% identical, at least 90% identical,at least 95% identical, and in some embodiments, at least 96%. 97%. 98%or 99% identical to a polynucleotide encoding a polypeptide comprisingan antibody, or fragment thereof, to a human FZD receptor describedherein.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence can include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence. In other words, to obtain a polynucleotide having a nucleotidesequence at least 95% identical to a reference nucleotide sequence, upto 5% of the nucleotides in the reference sequence can be deleted orsubstituted with another nucleotide, or a number of nucleotides up to 5%of the total nucleotides in the reference sequence can be inserted intothe reference sequence. These mutations of the reference sequence canoccur at the amino- or carboxy-terminal positions of the referencenucleotide sequence or anywhere between those terminal positions,interspersed either individually among nucleotides in the referencesequence or in one or more contiguous groups within the referencesequence.

The polynucleotide variants can contain alterations in the codingregions, non-coding regions, or both. In some embodiments thepolynucleotide variants contain alterations which produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded polypeptide. In some embodiments,nucleotide variants are produced by silent substitutions due to thedegeneracy of the genetic code. Polynucleotide variants can be producedfor a variety of reasons. e.g., to optimize codon expression for aparticular host (change codons in the human mRNA to those preferred by abacterial host such as E. coli).

Vectors and cells comprising the polynucleotides described herein arealso provided.

IV. METHODS OF USE AND PHARMACEUTICAL COMPOSITIONS

The FZD-binding agents (including polypeptides and antibodies) of theinvention are useful in a variety of applications including, but notlimited to, therapeutic treatment methods, such as the treatment ofcancer. In certain embodiments, the agents are useful for inhibiting Wntsignaling (e.g. canonical Wnt signaling), inhibiting tumor growth,inducing differentiation, reducing tumor volume, and/or reducing thetumorigenicity of a tumor. The methods of use may be in vitro, ex vivo,or in vivo methods. In certain embodiments, the FZD-binding agent orpolypeptide or antibody is an antagonist of the one or more humanfrizzled receptors to which it binds.

In certain embodiments, the FZD-binding agents or antagonists are usedin the treatment of a disease associated with Wnt signaling activation.In certain embodiments, the methods comprise administering an effectiveamount of the FZD-binding agent to a subject. In certain embodiments,the disease is cancer or polycystic kidney disease. In particularembodiments, the disease is a disease dependent upon Wnt signaling. Inparticular embodiments, the Wnt signaling is canonical Wnt signaling. Incertain embodiments, the FZD-binding agents or antagonists are used inthe treatment of disorders characterized by increased levels of stemcells and/or progenitor cells.

In certain embodiments, the disease treated with the FZD-binding agentor antagonist (e.g., an anti-FZD antibody) is a cancer. In certainembodiments, the cancer is characterized by Wnt-dependent tumors. Incertain embodiments, the cancer is characterized by tumors expressingone or frizzled receptors to which the FZD-binding agent (e.g.,antibody) binds. In certain embodiments, the cancer is characterized bytumors expressing one or more genes in a Wnt gene signature.

In certain embodiments, the disease treated with the FZD-binding agentor antagonist is not a cancer. For example, the disease may be ametabolic disorder such as obesity or diabetes (e.g., type II diabetes)(Jin T., Diabetologia, 2008 October; 51(10):1771-80). Alternatively, thedisease may be a bone disorder such as osteoporosis, osteoarthritis, orrheumatoid arthritis (Corr M., Nat Clin Pract Rheumatol, 2008 October;4(10):550-6: Day et al., Bone Joint Surg Am, 2008 February; 90 Suppl1:19-24). The disease may also be a kidney disorder, such as apolycystic kidney disease (Harris et al., Annu Rev Med, 2008 Oct. 23;Schmidt-Ott et al., Kidney Int, 2008 October; 74(8):1004-8; Benzing etal., J Am Soc Nephrol, 2007 May; 18(5): 1389-98). Alternatively, eyedisorders including, but not limited to, macular degeneration andfamilial exudative vitreoretinopathy may be treated (Lad et al., StemCells Dev, 2008 Aug. 8). Cardiovascular disorders, including myocardialinfarction, atherosclerosis, valve disorders, and cardiac hypertrophymay also be treated (Al-Aly Z., Transl Res, 2008 May; 151(5):233-9;Kobayashi et al., Nat Cell Biol, 2009 January; 11(1):46-55; van Gijn etal., Cardiovasc Res, 2002 July; 55(1):16-24; Christman et al., Am JPhysiol Heart Circ Physiol, 2008 June; 294(6):H2864-70; Malekar et al.,Hypertension, 2010 April; 55(4):939-45 (Epub 2010 Feb. 22).). In someembodiments, the disease is a pulmonary disorder such as idiopathicpulmonary arterial hypertension or pulmonary fibrosis (Laumanns et al.,Am J Respir Cell Mol Biol, 2008 Nov. 21; Kinigshoff et al., PLoS ONE,2008 May 14; 3(5):e2142). In some embodiments, the disease treated withthe FZD-binding agent is a liver disease, such as cirrhosis or liverfibrosis (Cheng et al., Am J Physiol Gastrointest Liver Physiol, 2008January; 294(1):G39-49).

Accordingly, the present invention provides for methods of treatingpolycystic kidney disease comprising administering to a subject in needthereof an effective amount of a FZD-binding agent, polypeptide, orantibody. In certain embodiments, the FZD-binding agent, polypeptide, orantibody binds to the BBS of a human FZD receptor (e.g., FZD8). Incertain embodiments, the FZD-binding polypeptide comprises a sequencehaving at least 80% identity with a sequence selected from the groupconsisting of SEQ ID NOs:98-111 and SEQ ID NOs:113-116. In certainalternative embodiments, the polypeptide comprises the sequence of SEQID NO: 112. In still further embodiments, the FZD-binding agent is 18R5.

The present invention provides for methods of treating cancer comprisingadministering a therapeutically effective amount of a FZD-binding agentto a subject (e.g. a subject in need of treatment). In certainembodiments, the cancer is a cancer selected from the group consistingof colorectal cancer, pancreatic cancer, lung cancer, ovarian cancer,liver cancer, breast cancer, kidney cancer, prostate cancer,gastrointestinal cancer, melanoma, cervical cancer, bladder cancer,glioblastoma, and head and neck cancer. In certain embodiments, thecancer is pancreatic cancer. In certain embodiments, the cancer iscolorectal cancer. In certain embodiments, the subject is a human.

The present invention further provides methods for inhibiting tumorgrowth using the antibodies or other agents described herein. In certainembodiments, the method of inhibiting the tumor growth comprisescontacting a tumor or tumor cells with an effective amount of aFZD-binding agent (e.g., an antibody or other polypeptide) in vitro. Forexample, an immortalized cell line or a cancer cell line that expressesthe targeted FZD(s) is cultured in medium to which is added the antibodyor other agent to inhibit tumor growth. In some embodiments, tumor cellsare isolated from a patient sample such as, for example, a tissuebiopsy, pleural effusion, or blood sample and cultured in medium towhich is added an FZD-binding agent to inhibit tumor growth.

In some embodiments, the method of inhibiting tumor growth comprisescontacting the tumor or tumor cells with an effective amount of theFZD-binding agent (e.g., antibody) in vivo. In certain embodiments,contacting a tumor or tumor cell with a FZD-binding agent is undertakenin an animal model. For example, FZD-binding agents may be administeredto xenografts expressing one or more FZDs that have been grown inimmunocompromised mice (e.g. NOD/SCID mice) to inhibit tumor growth. Insome embodiments, cancer stem cells are isolated from a patient samplesuch as, for example, a tissue biopsy, pleural effusion, or blood sampleand injected into immunocompromised mice that are then administered aFZD-binding agent to inhibit tumor cell growth. In some embodiments, theFZD-binding agent is administered at the same time or shortly afterintroduction of tumorigenic cells into the animal to prevent tumorgrowth. In some embodiments, the FZD-binding agent is administered as atherapeutic after the tumorigenic cells have grown to a specified size.

In certain embodiments, the method of inhibiting tumor growth comprisesadministering to a subject a therapeutically effective amount of aFZD-binding agent. In certain embodiments, the subject is a human. Incertain embodiments, the subject has a tumor or has had a tumor removed.

In certain embodiments, the tumor is a tumor in which Wnt signaling isactive. In certain embodiment, the Wnt signaling that is active iscanonical Wnt signaling. In certain embodiments, the tumor is aWnt-dependent tumor. For example, in some embodiments, the tumor issensitive to axin over-expression. In certain embodiments, the tumordoes not comprise an inactivating mutation (e.g., a truncating mutation)in the adenomatous polyposis coli (APC) tumor suppressor gene or anactivating mutation in the beta-catenin gene. In certain embodiments,the tumor expresses one or more genes in a Wnt gene signature. Incertain embodiments, the cancer for which a subject is being treatedinvolves such a tumor.

In certain embodiments, the tumor expresses the one or more humanfrizzled receptors to which the FZD-binding agent or antibody binds. Incertain embodiments, the tumor overexpresses the human frizzledreceptor(s).

In certain embodiments, the tumor is a tumor selected from the groupconsisting of colorectal tumor, pancreatic tumor, lung tumor, ovariantumor, liver tumor, breast tumor, kidney tumor, prostate tumor,gastrointestinal tumor, melanoma, cervical tumor, bladder tumor,glioblastoma, and head and neck tumor. In certain embodiments, the tumoris a colorectal tumor. In certain embodiments, the tumor is a pancreatictumor.

The invention also provides a method of inhibiting Wnt signaling in acell comprising contacting the cell with an effective amount of aFZD-binding agent. In certain embodiments, the cell is a tumor cell. Incertain embodiments, the method is an in vivo method wherein the step ofcontacting the cell with the agent comprises administering atherapeutically effective amount of the agent to the subject. In somealternative embodiments, the method is an in vitro or ex vivo method. Incertain embodiments, the Wnt signaling that is inhibited is canonicalWnt signaling. In certain embodiments, the Wnt signaling is signaling byWNT1, WNT2, WNT3, WNT3A. WNT7a, WNT7b, and/or WNT10B. In certainembodiments, the Wnt signaling is signaling by WNT1, WNT3A, WNT7b,and/or WNT 10B.

In addition, the invention provides a method of reducing thetumorigenicity of a tumor in a subject, comprising contacting the tumorwith an effective amount of a FZD-binding agent or administering atherapeutically effective amount of a FZD-binding agent to the subject.In certain embodiments, the tumor comprises cancer stem cells. Incertain embodiments, the frequency of cancer stem cells in the tumor isreduced by administration of the agent.

Thus, the invention also provides a method of reducing the frequency ofcancer stem cells in a tumor, comprising contacting the tumor with aneffective amount of a FZD-binding agent (e.g. an anti-FZD antibody orother FZD-binding polypeptide).

The invention further provides methods of differentiating tumorigeniccells into non-tumorigenic cells comprising contacting the tumorigeniccells with a FZD-binding agent (for example, by administering theFZD-binding agent to a subject that has a tumor comprising thetumorigenic cells or that has had such a tumor removed. In certainembodiments, the tumorigenic cells are pancreatic tumor cells. Incertain alternative embodiments, the tumorigenic cells are colon tumorcells.

The use of the FZD-binding agents, polypeptides, or antibodies describedherein to induce the differentiation of cells, including, but notlimited to tumor cells, is also provided. For example, methods ofinducing cells to differentiate comprising contacting the cells with aneffective amount of a FZD-binding agent (e.g., an anti-FZD antibody orother FZD-binding polypeptide) described herein are envisioned. Methodsof inducing cells in a tumor in a subject to differentiate comprisingadministering a therapeutically effective amount of a FZD-binding agent,polypeptide, or antibody to the subject are also provided. In certainembodiments, the tumor is a pancreatic tumor. In certain otherembodiments, the tumor is a colon tumor.

Methods of treating a disease or disorder in a subject, wherein thedisease or disorder is associated with Wnt signaling activation and/oris characterized by an increased level of stem cells and/or progenitorcells are further provided. In some embodiments, the treatment methodscomprise administering a therapeutically effective amount of theFZD-binding agent, polypeptide, or antibody to the subject. In certainembodiments, the Wnt signaling is canonical Wnt signaling.

The present invention further provides methods of reducingmyofibrolblast activation in the stroma of a solid tumor, comprisingcontacting the stroma with an effective amount of the FZD-binding agent,polypeptide or antibody.

The present invention further provides pharmaceutical compositionscomprising one or more of the FZD-binding agents described herein. Incertain embodiments, the pharmaceutical compositions further comprise apharmaceutically acceptable vehicle. These pharmaceutical compositionsfind use in inhibiting tumor growth and treating cancer in humanpatients.

In certain embodiments, formulations are prepared for storage and use bycombining a purified antibody or agent of the present invention with apharmaceutically acceptable vehicle (e.g. carrier, excipient)(Remington, The Science and Practice of Pharmacy 20th Edition MackPublishing. 2000). Suitable pharmaceutically acceptable vehiclesinclude, but are not limited to, nontoxic buffers such as phosphate,citrate, and other organic acids; salts such as sodium chloride;antioxidants including ascorbic acid and methionine; preservatives (e.g.octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride:benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens, such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight polypeptides (e.g. less than about 10 amino acid residues);proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilicpolymers such as polyvinylpyrrolidone; amino acids such as glycine,glutamine, asparagine, histidine, arginine, or lysine; carbohydratessuch as monosacchandes, disaccharides, glucose, mannose, or dextrins;chelating agents such as EDTA; sugars such as sucrose, mannitol,trehalose or sorbitol; salt-forming counter-ions such as sodium; metalcomplexes (e.g. Zn-protein complexes); and non-ionic surfactants such asTWEEN or polyethylene glycol (PEG).

In certain embodiments, the pharmaceutical composition is frozen. Incertain alternative embodiments, the pharmaceutical composition islyophilized.

The invention provides formulations for the antibodies described herein,such as 18R5, which comprise one or more (or all) of the followingcomponents: 30-50 mM Histidine, 70-110 mM Sodium Chloride, 60-100 mMSucrose, and/or 0.01%-0.04% Polysorbate-20, with a pH of 5.5 to 5.9. Incertain embodiments, the formulation comprises 40 mM Histidine, 86 mMSodium Chloride, 82 mM Sucrose, and 0.02% Polysorbate-20, pH 5.7. Thus,in one embodiment, an 18R5 formulation comprises 5-20 mg/mL 18R5, 40 mMHistidine, 86 mM Sodium Chloride, 82 mM Sucrose, and 0.02%Polysorbate-20, pH 5.7. In another embodiment, an 18R5 formulationcomprises 5-20 mg/mL 18R5, 56 mM Histidine, 86 mM Sodium Chloride, 82 mMSucrose, and 0.02% Polysorbate-20, pH 5.7. In certain embodiments, theconcentration of 18R5 in the formulation is 10 mg/mL. In certainembodiments, the pharmaceutical composition is frozen.

The pharmaceutical compositions of the present invention can beadministered in any number of ways for either local or systemictreatment. Administration can be topical (such as to mucous membranesincluding vaginal and rectal delivery) such as transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders; pulmonary (e.g., by inhalation or insufflation of powdersor aerosols, including by nebulizer: intratracheal, intranasal,epidermal and transdermal); oral; or parenteral including intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial (e.g., intrathecal or intraventricular)administration.

The therapeutic formulation can be in unit dosage form. Suchformulations include tablets, pills, capsules, powders, granules,solutions or suspensions in water or non-aqueous media, or suppositoriesfor oral, parenteral, or rectal administration or for administration byinhalation. In solid compositions such as tablets the principal activeingredient is mixed with a pharmaceutical carrier. Conventionaltableting ingredients include corn starch, lactose, sucrose, sorbitol,talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, andother diluents (e.g. water) to form a solid preformulation compositioncontaining a homogeneous mixture of a compound of the present invention,or a non-toxic pharmaceutically acceptable salt thereof. The solidpreformulation composition is then subdivided into unit dosage forms ofthe type described above. The tablets, pills, etc of the novelcomposition can be coated or otherwise compounded to provide a dosageform affording the advantage of prolonged action. For example, thetablet or pill can comprise an inner composition covered by an outercomponent. Furthermore, the two components can be separated by anenteric layer that serves to resist disintegration and permits the innercomponent to pass intact through the stomach or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol and cellulose acetate.

The antibodies or agents can also be entrapped in microcapsules. Suchmicrocapsules are prepared, for example, by coacervation techniques orby interfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions as described in Remington, TheScience and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

In certain embodiments, pharmaceutical formulations include antibodiesor other agents of the present invention complexed with liposomes(Epstein, et al., 1985, Proc. Natl. Acad. Sci. USA 82:3688; Hwang, etal., 1980, Proc. Natl. Acad. Sci. USA 77:4030; and U.S. Pat. Nos.4,485,045 and 4,544,545). Liposomes with enhanced circulation time aredisclosed in U.S. Pat. No. 5,013,556. Some liposomes can be generated bythe reverse phase evaporation with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter.

In addition sustained-release preparations can be prepared. Suitableexamples of sustained-release preparations include semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles (e.g. films, ormicrocapsules). Examples of sustained-release matrices includepolyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) orpoly(v nylalcohol), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and 7 ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), sucrose acetateisobutyrate, and poly-D-(−)-3-hydroxybutyric acid.

In certain embodiments, in addition to administering the FZD-bindingagent, the method or treatment further comprises administering a secondanti-cancer agent (prior to, concurrently with, and/or subsequently toadministration of the FZD-binding agent). Pharmaceutical compositionscomprising the FZD-binding agent and the second anti-cancer agent arealso provided. In certain embodiments, the administration of thecombination of the FZD-binding agent and a second anti-cancer agent hasa synergistic effect, such as a synergistic effect on the frequency ofcancer stem cells.

It will be appreciated that the combination of a FZD-binding agent and asecond anti-cancer agent may be administered in any order orconcurrently. In selected embodiments, the FZD-binding agents will beadministered to patients that have previously undergone treatment withthe second anti-cancer agent. In certain other embodiments, theFZD-binding agent and the second anti-cancer agent will be administeredsubstantially simultaneously or concurrently. For example, a subject maybe given the FZD-binding agent while undergoing a course of treatmentwith the second anti-cancer agent (e.g. chemotherapy). In certainembodiments, the FZD-binding agent will be administered within 1 year ofthe treatment with the second anti-cancer agent. In certain alternativeembodiments, the FZD-binding agent will be administered within 10, 8, 6,4, or 2 months of any treatment with the second anti-cancer agent. Incertain other embodiments, the FZD-binding agent will be administeredwithin 4, 3, 2, or 1 week of any treatment with the second anti-canceragent. In some embodiments, the FZD-binding agent will be administeredwithin 5, 4, 3, 2, or 1 days of any treatment with the secondanti-cancer agent. It will further be appreciated that the two agents ortreatment may be administered to the subject within a matter of hours orminutes (i.e., substantially simultaneously).

Useful classes of anti-cancer agents include, for example, antitubulinagents, auristatins, DNA minor groove binders, DNA replicationinhibitiors, alkylating agents (e.g., platinum complexes such ascis-platin, mono(platinum), bis(platinum) and tri-nuclear platinumcomplexes and carboplatin), anthracyclines, antibiotics antifolates,antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides,fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas,platinols, performing compounds, purine antimetabolites, puromycins,radiation sensitizers, steroids, taxanes, topoisomerase inhibitors,vinca alkaloids, or the like. In certain embodiments, the secondanti-cancer agent is an antimetabolite, an antimitotic, a topoisomeraseinhibitor, or an angiogenesis inhibitor.

Anticancer agents that may be administered in combination with theFZD-binding agents include chemotherapeutic agents. Thus, in someembodiments, the method or treatment involves the combinedadministration of an antibody or agent of the present invention and achemotherapeutic agent or cocktail of multiple differentchemotherapeutic agents. Treatment with an antibody can occur prior to,concurrently with, or subsequent to administration of chemotherapies.Chemotherapies contemplated by the invention include chemical substancesor drugs which are known in the art and are commercially available, suchas Gemcitabine, Irinotecan, Doxorubicin, 5-Fluorouracil, Cytosinearabinoside (“Ara-C”), Cyclophosphamide, Thiotepa, Busulfan, Cytoxin,TAXOL, Methotrexate, Cisplatin, Melphalan, Vinblastine and Carboplatin.Combined administration can include co-administration, either in asingle pharmaceutical formulation or using separate formulations, orconsecutive administration in either order but generally within a timeperiod such that all active agents can exert their biological activitiessimultaneously. Preparation and dosing schedules for suchchemotherapeutic agents can be used according to manufacturers'instructions or as determined empirically by the skilled practitioner.Preparation and dosing schedules for such chemotherapy are alsodescribed in Chemotherapy Service Ed. M. C. Perry, Williams & Wilkins,Baltimore, Md. (1992).

Chemotherapeutic agents useful in the instant invention also include,but are not limited to, alkylating agents such as thiotepa andcyclosphosphamide (CYTOXAN); alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethylencthiophosphaoramidc and trimethylolomelamime nitrogen mustardssuch as chlorambucil, chlornaphazine, cholophosphamide, estramustine,ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichin, phenesterine, prednimustine, trofosfamide,uracil mustard; nitrosureas such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, ranimustine; antibiotics such asaclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin,chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate: purine analogs such asfludarabine. 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane: folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate: etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK.; razoxane;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine: dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine:arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.paclitaxel (TAXOL. Bristol-Myers Squibb Oncology, Princeton, N.J.) anddoxetaxel (TAXOTERE, Rhone-Poulenc Rorer. Antony. France); chlorambucil;gemcitabine: 6-thioguanine; mercaptopurine; methotrexate; platinumanalogs such as cisplatin and carboplatin; vinblastine; platinum;etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;xeloda; ibandronate; CPT11: topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoic acid; esperamicins;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above. Chemotherapeutic agents also includeanti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens including for example tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston);and antiandrogens such as flutamide, nilutamide, bicalutamide,leuprolide, and goserelin; and pharmaceutically acceptable salts, acidsor derivatives of any of the above.

In certain embodiments, the chemotherapeutic agent is a topoisomeraseinhibitor. Topoisomerase inhibitors are chemotherapy agents thatinterfere with the action of a topoisomerase enzyme (e.g., topoisomeraseI or II). Topoisomerase inhibitors include, but are not limited to,doxorubicin HCL, daunorubicin citrate, mitoxantrone HCL, actinomycin D,etoposide, Topotecan HCL, teniposide (VM-26), and irinotecan. In certainembodiments, the second anticancer agent is irinotecan. In certainembodiments, the tumor to be treated is a colorectal tumor and thesecond anticancer agent is a topoisomerase inhibitor, such asirinotecan.

In certain embodiments, the chemotherapeutic agent is ananti-metabolite. An anti-metabolite is a chemical with a structure thatis similar to a metabolite required for normal biochemical reactions,yet different enough to interfere with one or more normal functions ofcells, such as cell division. Anti-metabolites include, but are notlimited to, gemcitabine, fluorouracil, capecitabine, methotrexatesodium, ralitrexed, Pemetrexed, tegafur, cytosine arabinoside,THIOGUANINE (GlaxoSmithKline), 5-azacytidine, 6-mercaptopurine,azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate, andcladribine, as well as pharmaceutically acceptable salts, acids, orderivatives of any of these. In certain embodiments, the secondanticancer agent is gemcitabine. In certain embodiments, the tumor to betreated is a pancreatic tumor and the second anticancer agent is ananti-metabolite (e.g., gemcitabine).

In certain embodiments, the chemotherapeutic agent is an antimitoticagent, including, but not limited to, agents that bind tubulin. By wayof non-limiting example, the agent comprises a taxane. In certainembodiments, the agent comprises paclitaxel or docetaxel, or apharmaceutically acceptable salt, acid, or derivative of paclitaxel ordocetaxel. In certain embodiments, the agent is paclitaxel (TAXOL),docetaxel (TAXOTERE), albumin-bound paclitaxel (e.g., ABRAXANE),DHA-paclitaxel, or PG-paclitaxel. In certain alternative embodiments,the antimitotic agent comprises a vinka alkaloid, such as vincristine,binblastine, vinorelbine, or vindesine, or pharmaceutically acceptablesalts, acids, or derivatives thereof. In some embodiments, theantimitotic agent is an inhibitor of Eg5 kinesin or an inhibitor of amitotic kinase such as Aurora A or Plk1. In certain embodiments wherethe chemotherapeutic agent administered in combination with theFZD-binding agent or polypeptide or antibody comprises an antimitoticagent, the cancer or tumor being treated is breast cancer or a breasttumor.

In certain embodiments, the treatment involves the combinedadministration of an antibody (or other agent) of the present inventionand radiation therapy. Treatment with the antibody (or agent) can occurprior to, concurrently with, or subsequent to administration ofradiation therapy. Any dosing schedules for such radiation therapy canbe used as determined by the skilled practitioner.

In some embodiments, the second anti-cancer agent comprises an antibody.Thus, treatment can involve the combined administration of antibodies(or other agents) of the present invention with other antibodies againstadditional tumor-associated antigens including, but not limited to,antibodies that bind to EGFR, ErbB2, HER2, DLL4, Notch and/or VEGF.Exemplary, anti-DLL4 antibodies, are described, for example, in U.S.Patent Application Publication No. US 2008/0187532, incorporated byreference herein in its entirety. In certain embodiments, the secondanti-cancer agent is an antibody that is an angiogenesis inhibitor(e.g., an anti-VEGF antibody). Additional anti-DLL4 antibodies aredescribed in, e.g., International Patent Publication Nos. WO 2008/091222and WO 2008/0793326, and U.S. Patent Application Publication Nos. US2008/0014196, US 2008/0175847, US 2008/0181899, and US 2008/0107648,each of which is incorporated by reference herein in its entirety.Exemplary anti-Notch antibodies, are described, for example, in U.S.Patent Application Publication No. US 2008/0131434, incorporated byreference herein in its entirety. In certain embodiments, the secondanti-cancer agent is an antibody that is an angiogenesis inhibitor(e.g., an anti-VEGF antibody). In certain embodiments, the secondanti-cancer agent is an inhibitor of Notch signaling. In certainembodiments, the second anti-cancer agent is AVASTIN (Bevacizumab),Herceptin (Trastuzumab), VECTIBIX (Panitumumab), or Erbitux (Cetuximab).Combined administration can include co-administration, either in asingle pharmaceutical formulation or using separate formulations, orconsecutive administration in either order but generally within a timeperiod such that all active agents can exert their biological activitiessimultaneously.

Furthermore, treatment can include administration of one or morecytokines (e.g., lymphokines, interleukins, tumor necrosis factors,and/or growth factors) or can be accompanied by surgical removal ofcancer cells or any other therapy deemed necessary by a treatingphysician.

For the treatment of the disease, the appropriate dosage of an antibodyor agent of the present invention depends on the type of disease to betreated, the severity and course of the disease, the responsiveness ofthe disease, whether the antibody or agent is administered fortherapeutic or preventative purposes, previous therapy, patient'sclinical history, and so on all at the discretion of the treatingphysician. The antibody or agent can be administered one time or over aseries of treatments lasting from several days to several months, oruntil a cure is effected or a diminution of the disease state isachieved (e.g. reduction in tumor size). Optimal dosing schedules can becalculated from measurements of drug accumulation in the body of thepatient and will vary depending on the relative potency of an individualantibody or agent. The administering physician can easily determineoptimum dosages, dosing methodologies and repetition rates. In certainembodiments, dosage is from 0.01 μg to 100 mg per kg of body weight, andcan be given once or more daily, weekly, monthly or yearly. In certainembodiments, the antibody or other FZD-binding agent is given once everytwo weeks or once every three weeks. In certain embodiments, the dosageof the antibody or other FZD-binding agent is from about 0.1 mg to about20 mg per kg of body weight. The treating physician can estimaterepetition rates for dosing based on measured residence times andconcentrations of the drug in bodily fluids or tissues.

V. WNT GENE SIGNATURE AND USES THEREOF

The present invention further provides a Wnt gene signature, a genesignature indicative of Wnt signaling activity in tumors, which may beused in the selection of tumors, patients, and/or therapy.

The Wnt gene signature comprises the differential expression of a set ofgenes in tumors in which Wnt signaling is activated (and/or which aredependent upon Wnt signaling), relative to tumors in which Wnt signalingis not activated. In certain embodiments, the Wnt signaling is canonicalWnt signaling. The Wnt gene signature is useful for the identificationof tumors and/or patients likely to respond to treatment with aninhibitor of Wnt signaling (e.g. a FZD-binding agent that is anantagonist of at least one human frizzled receptor and/or an inhibitorof Wnt signaling).

In certain embodiments, the Wnt gene signature comprises one or moregenes (i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, or 19 genes) listed in Table 3, below. The “Probe Set ID” numbersare the probe set ID numbers for the GeneChip® Human Genome U133 Plus2.0 Array (“HG_UI33_Plus_(—)2”; Affymetrix, Santa Clara, Calif.). Intumors in which Wnt signaling is active (i.e., tumors which are positivefor a Wnt gene signature), the expression level(s) of the gene(s) inTable 3 that comprise the Wnt gene signature are elevated relative totumors in which Wnt signaling is not active. In some embodiments, theWnt gene signature comprises two or more genes listed in Table 3, below.In some embodiments, the Wnt gene signature comprises three or more,four or more, five or more, six or more, seven or more, eight or more,nine or more, ten or more, eleven or more, twelve or more, thirteen ormore, fourteen or more, fifteen or more, sixteen or more, seventeen ormore, eighteen or more, or nineteen of the genes listed in Table 3,below. In certain embodiments, the tumor being assessed for expressionlevels of the one or more genes in Table 3 is a colorectal tumor. Incertain embodiments, the Wnt gene signature comprises AXIN2 and/orFOXQ1. In certain embodiments, the tumor is a colorectal tumor and theWnt gene signature comprises AXIN2, LGR5, and/or FOXQ1.

TABLE 3 Exemplary Wnt gene signature genes HG_U133_Plus_2 Gene Probe SetID Symbol Description 222696_at AXIN2 axin 2 (conductin, axil)206286_s_at TDGF1 teratocarcinoma-derived growth factor 1 213880_at LGR5leucine-rich repeal-containing G protein-coupled receptor 5 207217_s_atNOX1 NADPH oxidase 1 209588_at EPHB2 EPH receptor B2 212850_s_at LRP4low density lipoprotein receptor-related protein 4 205107_s_at EFNA4ephrin-A4 214058_at MYCL1 v-myc myelocytomatosis viral oncogene homolog1, lung carcinoma derived (avian) 209864_at FRAT2 frequently rearrangedin advanced T-cell lymphomas 2 208121_s_at PTPRO protein tyrosinephosphatase, receptor type, O 229376_at unknown expressed sequence tag(EST) 202431_s_at MYC v-myc myelocytomatosis viral oncogene homolog(avian) 212444_at GPCR5A G protein-coupled receptor, family C, group 5,member A 222938_x_at ENPP3 ectonucleotidepyrophosphatase/phosphodiesterase 3 241607_at LOC730102 hypotheticalprotein LOC730102 227475_at FOXQ1 forkhead box Q1 230398_at TNS4 tensin4 40284_at FOXA2 forkhead box A2 219704_at YBX2 Y box binding protein 2

Methods of using the Wnt gene signature to select patients (or toidentify a patient) suitable for treatment with a Wnt pathway inhibitoror for assessing the efficacy of a particular therapy are also provided.In certain embodiments, the Wnt signaling inhibitor is a FZD-bindingagent, such as an antagonistic FZD antibody. For example, a patient maybe identified as being suitable for treatment with a FZD-binding agent(or FZD-binding agents) by determining whether a tumor in the patient orthat has been removed from the patient exhibits a Wnt gene signature. Incertain embodiments, detecting the Wnt gene signature comprisesassessing the expression level of one or more genes in Table 3 in thetumor. If expression levels of the one or more genes in Table 3 thatcomprise the Wnt gene signature are elevated in the tumor (thusindicating that Wnt signaling is active in the tumor), the patient isidentified as being suitable for treatment with a FZD-binding agent,such as an anti-FZD antibody that inhibits Wnt signaling. Methods ofusing the Wnt gene signature to select a suitable therapy for aparticular patient are likewise provided.

The invention provides a method of treating cancer in a patient having atumor or from whom a tumor has been removed, comprising (a) providingthe expression level of one or more genes in Table 3 in the tumor (b)selecting the patient for beginning or continuing treatment with aFZD-binding agent based on the expression level of the one or moregenes, and (c) administering the FZD-binding agent to the patient. Incertain embodiments, the method comprises measuring the expression levelof the one or more genes in the tumor. In certain embodiments, theexpression level of the one or more genes is compared to a control orreference level.

Methods of identifying tumor which may be responsive to treatment withan inhibitor of Wnt signaling are also provided. In certain embodiments,the inhibitor of Wnt signaling is a FZD-binding agent. In certainembodiments, the methods comprise testing the tumor for a Wnt genesignature. In certain embodiments, the methods comprise assessing theexpression level of one or more genes in Table 3 in the tumor.

Methods of screening drug candidates against tumors identified asexhibiting the Wnt gene signature are also provided. In certainembodiments, the drug candidates are Wnt signaling inhibitors. Such drugcandidates are preferably tested for efficacy on those tumors in whichWnt signaling is active and/or which are dependent upon Wnt signaling.

The present invention also provides a method of screening a drugcandidate comprising assessing the expression level of one or more genesin Table 3 in a tumor (b) selecting the tumor for testing with the drugcandidate based (at least in part) on the expression level of the one ormore genes, and (c) testing the effect of the drug candidate on thetumor.

In addition, in certain embodiments, the effect of a drug on the Wntgene signature may be determined and used to assess the efficacy of atreatment of a tumor in which Wnt signaling is active. In certainembodiments, this provides a method of monitoring treatment of apatient. In some alternative embodiments, this provides a method ofassessing the efficacy of a drug candidate. In certain embodiments, adecrease in the expression levels of one or more genes in Table 3 (i.e.,a reduction in or elimination of the Wnt gene signature) indicatesefficacy of the treatment.

In certain embodiments, assessing the level of one or more genes in aWnt gene signature comprises determining the expression levels ofpolynucleotides of the one or more genes. In certain embodiments,detecting a Wnt gene signature comprises detecting mRNA expression ofpolynucleotides of the one or more genes that comprise the signature. Insome embodiments, the detection of mRNA expression is via Northern blot.In some embodiments, the detection of mRNA expression is via RT-PCR,real-time PCR or quantitative PCR using primer sets that specificallyamplify the polynucleotides comprising the cancer stem cell signature.In certain embodiments, the detection of mRNA comprises exposing asample to nucleic acid probes complementary to polynucleotidescomprising a cancer stem cell gene signature. In some embodiments, themRNA of the sample is converted to cDNA prior to detection. In someembodiments, the detection of mRNA is via microarrays that comprisepolynucleotides that hybridize to one or more genes in the Wnt genesignature.

In certain embodiments, assessing the level of one or more genes in aWnt gene signature comprises detecting polypeptides encoded by the oneor more genes. In some embodiments, the assessment of levels of thepolypeptide expression products of the one or more genes comprisesexposing a sample to antibodies specific to the polypeptides anddetecting the binding of the antibodies to the polypeptides by, forexample, quantitative immunofluorescence, or ELISA. Other detectionmeans are known to one of ordinary skill in the art see e.g., U.S. Pat.No. 6,057,105.

An array comprising polynucleotides that hybridize under stringentconditions to one or more genes in Table 3 are also provided. A kitcomprising the array is also provided.

Kits comprising antibodies that bind the expression product of one ormore genes in Table 3 are also provided.

VI. KITS COMPRISING FZD-BINDING AGENTS

The present invention provides kits that comprise the antibodies orother agents described herein and that can be used to perform themethods described herein. In certain embodiments, a kit comprises atleast one purified antibody against one or more human frizzled receptorsin one or more containers. In some embodiments, the kits contain all ofthe components necessary and/or sufficient to perform a detection assay,including all controls, directions for performing assays, and anynecessary software for analysis and presentation of results. One skilledin the art will readily recognize that the disclosed antibodies oragents of the present invention can be readily incorporated into one ofthe established kit formats which are well known in the art.

Further provided are kits comprising a FZD-binding agent (e.g., aFZD-binding antibody), as well as a second anti-cancer agent. In certainembodiments, the second anti-cancer agent is a chemotherapeutic agent(e.g., gemcitabine or irinotecan). In certain embodiments, the secondanti-cancer agent is an angiogenesis inhibitor. In certain embodiments,the second anti-cancer agent is an inhibitor of Notch signaling (e.g.,an anti-DLL4 or anti-Notch antibody).

Also provided are kits comprising a FZD-binding agent and a reagent orreagents for assessing the expression of one or more gene in Table 3,above (“Exemplary Wnt gene signature genes”).

Embodiments of the present disclosure can be further defined byreference to the following non-limiting examples, which describe indetail preparation of certain antibodies of the present disclosure andmethods for using antibodies of the present disclosure. It will beapparent to those skilled in the art that many modifications, both tomaterials and methods, may be practiced without departing from the scopeof the present disclosure.

EXAMPLES

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application.

Example 1 Identification/Generation of Anti-FZD Antibodies

Human antibodies that specifically recognize one or more human Frizzledreceptors can be isolated using phage display. For example, a syntheticantibody library containing human antibody variable domains may bepanned for specific and high affinity recognition of the extracellulardomain of the human FZD7 receptor. Once a specific Fab with the desiredcharacteristics has been identified, the human variable regions of theFab are then cloned into an Ig expression vector containing human IgG2heavy-chain and light-chain (kappa or lambda) for expression of humanantibodies in CHO cells.

Phage display was used to identify a specific Fab, 18R8, that binds tothe extracellular domain of FZD7. 2×10¹³ Fab displaying phage particlesfrom a human Fab phage library were incubated with passively immobilizedrecombinant FZD7 ECD Fc protein. The non-specific phage were washed off,and then specific phage were eluted with DTT. The eluted output was usedto infect TG1 F+ bacteria, rescued with helper phage. Fab display wasthen induced with IPTG (0.25 mM). The output of this rescued round oneserved as the starting point for further selection rounds. Theselections were continued to round 3, and then the output was screenedin ELISA for specific Fabs to recombinant FZD7 ECD Fc protein. A Fabthat specifically bound to human FZD7 was identified.

The sequences of the variable regions of the identified Fab wereobtained. An N-linked glycosylation site was removed from the parentsequence through site-directed mutagenesis. The N-linked glycosylationsite. Asn, in the heavy chain CDR1 was changed to His. This mutation wasmade to prevent glycosylation during expression in mammalian systems.The resulting Fab was designated 18R8. The heavy chain and light chainCDR sequences of 18R8 are shown below in Table 4, below. The VH and VLsequences of 18R8 are provided in SEQ ID NO: 10 and SEQ ID NO: 12,respectively.

TABLE 4 CDRs of 18R8 and 18R5 human antibodies Heavy Chain Light ChainLead CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 18R8 GFTFSHYTLS VISGDGSYTYYADSVKGNFIKYVFAN SGDKLGKKYAS EKDNRPSG SSFAGNSLE (SEQ ID NO: 1) (SEQ ID NO: 2)(SEQ ID NO: 3) (SEQ ID NO: 4) (SEQ. ID NO: 5) (SEQ ID NO: 6) 18R5GFTFSHYTLS VISGDGSYTYYADSVKG NFIKYVFAN SGDNIGSFYVH DKSNRPSG QSYANTLSL(SEQ ID NO: 1) (SEQ. ID NO: 2) (SEQ ID NO: 3) (SEQ ID NO: 7)(SEQ ID NO: 8) (SEQ ID NO: 9) *Site directed change to remove N-linkedglycosylation site is underlined.

Anti-FZD Fab 18R5 was generated by associating the VH-CH1 chains of18R8Fab with a variety of VL-CL chains from the original Fab phagelibrary from which 18R8 was identified. 18R5 was isolated from thelibrary after three rounds of panning with immobilized recombinant FZD7ECD Fc protein. The sequences of the CDRs of 18R5 are shown in Table 4,above. The VL of the 18R5 antibody has the sequence shown in SEQ IDNO:14. The heavy chain CDRs and the VH of the 18R5 antibody areidentical to that of the 18R8 antibody.

The human variable regions of the 18R8 and 18R5Fabs were cloned into Igexpression vector containing human IgG2 heavy-chain and light-chain(lambda) for expression in CHO cells. The amino acid sequence of theheavy chain and light chain of the 18R8 IgG antibody (including signalsequences) are provided in SEQ ID NO: 11 and SEQ ID NO: 13,respectively. The signal sequence at the N-terminus of the amino acidsequence of each of the chains is cleaved upon secretion. The nucleicacid sequences encoding the heavy and light chains of the 18R8 IgGantibody are provided in SEQ ID NO:18 and SEQ ID NO:20, respectively.The amino acid sequence of the heavy chain and light chain of the 18R5IgG antibody are provided in SEQ ID NO:11 and SEQ ID NO:15,respectively. (Again, the signal sequence at the N-terminus of the aminoacid sequence of each of the chains is cleaved upon secretion.) Thenucleic acid sequences encoding the heavy and light chains of the 18R5IgG antibody are provided in SEQ ID NO:18 and SEQ ID NO:22,respectively. Protein A purification was used to purify the antibodies.

The K_(D)s of 18R8 and 18R5 antibodies were determined using the Biacore2000 system from Biacore Lifescience (GE Healthcare). Specifically,purified anti-Fzd7 antibodies were serially diluted in 2-fold incrementsfrom 100 to 0.78 nM in HBS-P (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.005%v/v Surfactant P20). Each dilution was tested against recombinant Fzd Fcproteins immobilized onto a CM5 Biacore chip. The association anddissociation rates were measured and K, values determined using theBiaevaluation software program (Table 5, below).

TABLE 5 Affinity of 18R8 and 18R5 IgG antibodies 18R8 18R5 FZD K_(D)(nM) K_(D) (nM) 1 15.6 4.6 2 6.2 3.3 5 4.2 1.9 7 5.2 1.8 8 29.3 5.8

Another anti-FZD antibody, 18R0512, was also prepared.

Example 2 FACS Analysis of Anti-FZD Antibodies Demonstrates Binding toMultiple Cell-Surface Human FZDs

Flow cytometry analysis was utilized to determine the ability ofantibodies to bind to cell surface expressed FZD proteins.

To enable robust cell surface expression of selected FZD proteins,mammalian expression plasmids comprising a CMV promoter upstream ofpolynucleotides that encode FZD were generated using standardrecombinant DNA technology (such constructs were termed “FL no FLAG”).Similar expression plasmids were generated for each of the ten humanfrizzled proteins. Alternative versions of the FZD expression vectorswere also prepared in which polynucleotides encoding an N-terminalsignal sequence-FLAG epitope tag fused to the N-terminus of the matureFZD protein were also generated by standard recombinant technology (suchconstructs were termed “FL flag”). Additionally, expression plasmidswere designed which encoded chimeric proteins comprised of either theCRD domain (also referred to as the “fri” domain) of the FZD or theentire N-terminal extracellular domain of the FZD protein fused to anN-terminal signal sequence-FLAG epitope (termed “fri flag” and “ECDflag” respectively), as well as a C-terminal section encoding thetransmembrane and cytoplasmic domain of human CD4 protein.

To measure antibody binding to FZD by flow cytometry HEK293 cells wereco-transfected with FZD expression vectors and the transfection markerGFP. Twenty-four to forty-eight hours post-transfection, cells werecollected in suspension and incubated on ice with anti-FZD antibodies(10 μg/ml unless otherwise indicated) or control IgG to detectbackground antibody binding. The cells were washed and primaryantibodies detected with Fc domain-specific secondary antibodiesconjugated to a fluorescent chromophore (e.g. phycoerythrin conjugatedanti-human IgG). Labeled cells were then analyzed by flow cytometry toidentify anti-FZD antibodies that specifically recognize cell surfaceexpression of FZD protein. Monoclonal antibodies 18R5 and 18R8recognized FZD on transfected cells. As shown in FIG. 1 and FIG. 2, both18R8 and 18R5 bind to multiple FZD including FZD1, FZD2, FZD5, FZD7, andFZD8. To examine the relative ability of 18R8 and 18R5 to bind to eachFZD protein, titration analysis was conducted wherein the amount ofantibody in the binding reaction was varied (FIG. 2). This analysisdemonstrated that 18R5 displayed greater binding potency to each of theFZD receptors (FZD1, FZD2, FZD5. FZD7, and FZD8) than 18R8.

Example 3 Inhibition of Wnt signaling by 18R8 and 18R5

The ability of the anti-FZD IgG antibodies 18R8 and 18R5 to blockactivation of the Wnt signaling pathway was determined in vitro usingluciferase reporter assays.

STF293 cells were cultured in DMEM supplemented with antibiotics and 10%FCS. The STF293 cells are 293 cells in which the following have beenstably integrated: (1) an 8xTCF Luc reporter vector containing sevencopies of the TCF binding site linked to a promoter upstream of afirefly luciferase reporter gene to measure canonical Wnt signalinglevels (Gazit et al., 1999, Oncogene 18:5959-66) and (2) a Renillaluciferase reporter (Promega; Madison, Wis.) as an internal control fortransfection efficiency. The cells were added to cultures plates. TheFZD antibodies to be tested (or no antibodies) were added. The cellswere then incubated in the presence or absence of Wnt3A-conditionedmedium that had been prepared from L cells that stably express Wnt3a(ATCC CRL-2647) or control conditioned media from L cells notoverexpressing Wnt3A (ATCC cell line CRL-2648). After overnightincubation, luciferase levels were measured using a dual luciferaseassay kit (Promega; Madison, Wis.) with firefly luciferase activitynormalized to Renilla luciferase activity.

The ability of the 18R8 and 18R5 antibodies to inhibit Wnt-inducedpathway activation was thus determined. STF293 cells were treated withdifferent concentrations of 18R8 or 18R5 IgG antibodies andWnt3A-conditioned medium was added. The cells were assayed 18 hourslater using the dual luciferase assay kit. The results are shown in FIG.3. Greater inhibition of the TCF signaling of the Wnt3a pathway wasobserved with the anti-FZD antibody 18R5.

In further experiments, the ability of the 18R8 antibody to antagonizesignaling by different Wnt ligands was determined. HEK 293 cells weretransfected with Wnt1, Wnt2, Wnt2b2, Wnt3, Wnt3a, Wnt7a, Wnt7b, Wnt8a,Wnt9b, and Wnt10b for forty eight hours by Fugene 6 (Roche). Wnt3Aconditioned medium (“WNT3ACM”) was used as a positive control ofactivation. STF293 cells, were cultured in DMEM supplemented withantibiotics and 10% FCS and treated with 20 μg/ml 18R8 antibody or noantibody. The Wnt-overexpressing HEK293 cells were then added. Eighteenhours following treatment, luciferase levels were measured using a dualluciferase assay kit. The results are shown in FIG. 4. The anti-FZDantibody 18R8 was shown to inhibit signaling of a variety of Wnts,including inhibiting Wnt1, Wnt2, Wnt3, Wnt7A, Wnt7B, and Wnt10B, inaddition to Wnt3A.

In similar experiments, the ability of the 18R5 antibody to inhibitsignaling by different Wnt ligands was determined. The results are shownin FIG. 67. 18R5 was found to inhibit beta-catenin signaling in responseto each of the Wnt family members that was active in the cell-basedreporter assay.

Example 4 18R8 Blocks Binding of FZD to Wnt

To assess the ability of 18R8 to block the binding of FZD to Wnt, 2 μlof 1.32 μg/μl soluble FZD8-Fc containing the Fri domain (amino acids1-157 of FZD8 linked in-frame to human IgG1 Fe) was added to culturemedium to bind Wnt3A (added as 20 μl of Wnt3A conditioned medium) eitheralone or in the presence of the 18R8 IgG antibody (added as 4 μl of 3.71μg/μl). The mixtures were incubated either alone or in the presence ofProtein A sepharose beads (GE Healthcare products; 20 μl of 50% solutionin PBS) for two his at 4° C. After the incubation, the protein A beads(and any proteins complexed to the protein A beads) in each sample wereremoved by spinning and the supernatant was assayed for ability toinduce 8xTCF luciferase activity. The supernatant was added to STF293cells, which stably express 8xTCF (eight copies of the TCF-bindingdomain upstream of a firefly luciferase reporter gene) to measurecanonical Wnt signaling levels and which were cultured in DMEMsupplemented with antibiotics and 10% FCS. Eighteen hours followingtreatment of the STF293 cells, luciferase levels were measured using adual luciferase assay kit (Promega; Madison, Wis.).

As seen in FIG. 5, Wnt 3A plus Protein A induces a strong activation ofthe reporter gene as measured by luciferase units (RLU) (FIG. 5, column1). Addition of Fzd8-fc induces a complex between Wnt 3A-Fzd8 and the Fcfusion protein with the Protein A sepharose beads, and once the ProteinA-Wnt3A-Fzd8 complex is removed by centrifugation, the resultingsupernatant is no longer capable of activating the reporter gene (FIG.5, column 2). Upon addition of 18R8, this complex is presumablydisrupted by the blockade of the antibody upon the Wnt3A interactingdomain of Fzd8, because although the Protein A-Fzd8-fc complex isremoved along with 18R8 which can readily interact with Protein Athrough its own Fc portion, reporter activity can be readily observed,suggesting sufficient levels of uncomplexed Wnt 3A (FIG. 5, column 3).18R8 also functions on the endogenous Fzd's present on the STF293 cells,because failure to remove 18R8 by Protein A does not show the return ofWnt 3A activation (FIG. 5, column 6).

The data in FIG. 5 show that the presence of the 18R8 IgG antibody inthe incubation reaction resulted in retention of Wnt3A activity withinthe supernatant relative to the activity observed with FZD8-Fc alone.These results indicate that 18R8 blocked the ability of FZD8 to bind toWnt3A and indicate that antibody binding to the epitope recognized by18R8 is able to block Wnt-FZD interactions.

Similarly, to determine whether 18R5 directly blocked the ability of Wntto interact with Fzd, additional binding studies were conducted. Theresults are shown in FIG. 68. Wnt3A, FZD5-Fc and 18R5 were co-incubatedas indicated, and then 18R5 and FZD5-Fc were removed by protein A IP.Supernatant was then assayed for WNT activity in an 8xTCF luciferasereporter assay. 18R5 blocked Wnt3A binding to FzdS-Fc, suggesting that amechanism by which 18R5 inhibits Wnt signaling involves the directinhibition of Wnt binding.

Example 5 Epitope Mapping of 18R8 and 18R5

To identify the FZD epitope recognized by the 18R8 IgG antibody, epitopemapping was performed. Flow cytometry analysis of cell surface expressedFZD was utilized to measure antibody binding. Mammalian expressionplasmid vectors comprising a CMV promoter upstream of polynucleotidesthat encode an N-terminal signal sequence FLAG epitope tag fused to theN-terminus of the Fri domain of FZD8 in turn fused to the transmembranedomain and intracellular domain of CD4 protein were generated bystandard recombinant technology. This expression construct allowsexpression of the FZD8 Fri domain on the cell surface, as well asexpression of a FLAG epitope tag to monitor expression. Site-directmutagenesis was then used to modify selected amino acids within theextracellular domain of FZD. HEK293 cells were co-transfected withexpression vectors encoding the FZD and the transfection marker GFP.Forty-eight hours post-transfection, cells were collected in suspensionand incubated on ice with anti-FZD antibody or control IgG to detectbackground antibody binding. The cells were washed and primaryantibodies detected with anti-antibody secondary antibodies conjugatedto a fluorescent chromophore. Labeled cells were then analyzed by flowcytometry to measure the binding of anti-FZD antibody to cell surfaceFZD.

In this manner, specific amino acids within the FZD extracellular domainthat were important for the binding of anti-FZD antibodies wereidentified. When amino acid residues 82-83 of FZD8 were mutated from PDto SQ, binding of the FZD8 by 18R8 was largely unaffected (FIG. 6).Similarly, when amino acid 109S was changed to 109Q, no appreciableeffect on binding was observed. On the other hand, when residues 70-71of FZD8 were changed from HQ to AE, binding of the FZD8 on cells by 18R8was clearly diminished (FIG. 6 and FIG. 7). When amino acids 66-67 weremutated from GL to AA, or when amino acids 68-69 were mutated from EV toQL, or when amino acids 126-127 were mutated from FG to NV, binding ofthe 18R8 antibody to FZD8 on the cells was similarly lost. The loss ofbinding to FZD when certain amino acids had been substituted theextracellular domain of FZD revealed specific recognition sites of theantibody. Thus, the antibody 18R8 was determined to bind to an epitopecomprising amino acids 66-71 GLEVHQ (SEQ ID NO:25) of FZD8 and aminoacids 124-128 GF of human FZD8. This epitope region is well conservedacross the human frizzled receptors to which FZD8 binds (i.e., FZD1,FZD2, FZD5. FZD7, and FZD8), and not highly conserved in those humanfrizzled receptors to which FZD8 does not bind (i.e., FZD3, FZD4, FZD6,FZD9, or FZD10).

FACS experiments comparing the binding of 18R5 IgG and 18R8 IgG to thewild-type and mutant FZD8 on cells were also performed. Theseexperiments demonstrated that the 18R8 antibody and the 18R5 antibodybind to a similar epitope on FZD8 (FIG. 7).

Example 6 Identification of the Biological Binding Site (BBS of the FZDReceptors)

The discovery of antibodies that inhibit Wnt signaling and the discoveryof the epitope within the FZD protein bound by these antibodies has nowenabled the analysis of which regions of the FZD protein structure areimportant for Wnt signaling. To examine this, the crystal structure ofthe Fri domain of mouse Fzd 8 was examined. We identified the bindingepitope of 18R8 and 18R5 as lying within a region of the FZD structurefor which a specific functional role had not previously beenappreciated. Moreover the epitope contained two separate surfaceelements of the Fzd (which we termed “top edge” and “bottom edge”)separated by a cleft. It was also discovered upon comparison of the tenhuman frizzled receptors that there was striking conservation of theidentity of amino acids that lined the bottom of this cleft. The regioncomprising this cleft, as well as the “top edge” and “bottom edge” towhich 18R8 and 18R5 bind has been designated the Biological Binding Site(BBS) of FZD. Shown in FIG. 9 are images of the structure of a Fzd Fridomain based on analysis of the previously reported crystal structure ofmouse FZD8 (Dann C E et al., Nature 412 (6842) 86-90, 2001) and analysisdone using the software program Pymol. Shown in the upper left image isa surface view of the FZD Fri domain with the region of the Fzd proteincomprising the biological binding site (BBS) indicated by a whitecircle. The regions designated as the “top edge”, “bottom edge” and the“cleft” of the BBS are each highlighted in darker surface coloration inseparate images at the bottom of the panel. The upper right image ofFIG. 9 highlights in a darker surface the residues that are conserved in9 or 10 of ten human Fzd family members.

Example 7 Inhibition of tumor growth in vivo by 18R5 Prevention ofWnt-dependent Tumor Growth by 18R5

Female NOD/SCID mice were injected at age 5-7 weeks with 50,000 mousemammary tumor virus (MMTV)-WNT1 tumor derived cells in the upper rightmammary fat pad. Transgenic (MMTV)-Wnt-1 mice exhibit discrete steps ofmammary tumorigenesis, including hyperplasia, invasive ductal carcinoma,and distant metastasis, and thus this mouse model of breast cancerprovides a useful tool for analyzing the role of Wnts in tumor formationand growth (Nusse and Varmus (1982) Cell 31:99-109). Tumors from thesemice were dissociated and these dissociated tumor cells used for tumorpropagation purposes. Mice with tumor cells implanted in the mammary fatpad were monitored twice a week. Once tumors were palpable, tumors weremeasured twice weekly and tumor volume was determined using the formula½(a×b²); where a=length, and b=breadth. Data are expressed as mean andmean±S.E.M. Group means were compared using Student's two-tailed,unpaired t test. Probability (p) values of <0.05 were interpreted assignificantly different. On day 19, mice with average tumor volume of 44mm³ were randomized into 2 groups of 10 animals each. Animals wereinjected with either control antibody, or 18R5 IgG antibody (10 mg/kg).Administration of the antibodies was performed via injection into theintra-peritoneal cavity, twice weekly. Treatment with the antibody 18R5completely abolished tumor growth as compared to tumors treated withcontrol antibody (FIG. 10; p=0.002).

Reduction of OMP-C28 Xenograft Tumor Growth by Combination Treatment of18RS and Irinotecan

In another embodiment anti-FZD antibodies were analyzed for theirability to reduce the growth of OMP-C28 colon tumor xenografts.Dissociated human OMP-C28 cells (10,000 per animal) were injectedsubcutaneously into 6-8 week old male NOD/SCID mice. Tumor growth wasmonitored weekly and tumor measurements were initiated once tumors werepalpable. On day 24 mice with average tumor volume of 129 mm³ wererandomized into 4 groups of 10 animals each. Animals were injected witheither control antibody, or 18R5 IgG antibody (10 mg/kg), or irinotecan(7.5 mg/kg) or combination of both 18R5 and irinotecan. Administrationof the antibodies and irinotecan was performed via injection into theintra-peritoneal cavity, twice weekly. Tumors were measured twice a weekand tumor volume was determined using the formula ½(a×b²); wherea=length, and b=breadth. Data are expressed as mean and mean±S.E.M.Group means were compared using Student's two-tailed, unpaired t test.Probability (p) values of <0.05 were interpreted as significantlydifferent. Treatment with 18R5 resulted in a 40% reduction in tumorgrowth, as shown in FIG. 11 (p=0.02). Furthermore, treatment with 18R5and irinotecan resulted in a 53% reduction of tumor growth relative totreatment with irinotecan alone (p=0.0002 vs. irinotecan alone) (FIG.11). Thus, 18R5 demonstrated anti-tumor growth activity in OMP-C28 colontumor model as a single agent as well as in combination with irinotecan.

Reduction of OMP-Pn4 Xenograft Tumor Growth by Combination Treatment of18R5 and Gemcitabine

In another embodiment, anti-FZD antibodies were analyzed for theirability to reduce the growth of OMP-Pn4 pancreatic tumor xenografts.NOD/SCID mice were purchased from Harlan (Indianapolis. Ind.) andallowed to acclimate for several days prior to the studies. Theestablishment and characterization of in vivo cancer stem cell-drivenpancreas xenograft models were described previously (Li et al., CancerRes., 67:1030-7, 2007). For efficacy studies. OMP-Pn4 human pancreatictumor cells were dissociated into single cell suspensions, resuspendedin 1:1 (v/v) mixture of FACS buffer (Hank's balanced salt solution[HBSS] supplemented with 2% heat-inactivated fetal bovine serum and 20mM Hepes) and Matrigel (BD Bioscience, San Jose, Calif.) and implantedsubcutaneously into the right flank region of 6-7 weeks old maleNOD/SCID mice with a 25-gauge needle containing 50,000 cells/100 μL.Tumor growth was monitored weekly and tumor measurements were initiatedonce tumors were palpable. At day 36, the mean tumor volumes reachedabout 120 mm³ and the tumor-bearing animals were randomized (4 groups of9 per group). Treatment was initiated two days later. Animals wereinjected with control antibody, with 18R5 IgG antibody (10 mg/kg), withgemcitabine (40 mg/kg), or with a combination of both 18R5 IgG antibodyand gemcitabine. Administration of the antibodies and/or gemcitabine wasperformed via injection into the intra-peritoneal cavity, once weekly.Tumor growth was measured by with an electronic caliper (Coast ToolsCompany, San Leandro, Calif.). Tumors were measured once a week andtumor volume was determined using the formula ½(a×b²); where a=length(longest axis of the tumor), and b=breadth (shortest axis of the tumor).Animals were weighed every day if they showed more than 15% body weightloss and euthanized if they showed 20% body weight. Data are expressedas mean±S.E.M. Differences in mean values between groups were analyzedby non-parimetric t test. Multiple comparisons used one-way ANOVA testwith posthoc t test comparison. Differences of p<0.05 are consideredsignificantly different. Software for statistical analysis was byGraphPad Prism4 (GraphPad Software Inc., San Diego, Calif.). At the endof the study, the mice were euthanized using a CO₂ chamber followed bycervical dislocation. Tumors were collected for RNA and histologicanalysis. The remaining tumors were transferred into cold medium 199 forprocessing into single cell suspensions for analysis of cancer stem cellfrequency.

The results of the OMP-Pn4 xenograft study are shown in FIG. 12.Treatment with the 18R5 antibody as monotherapy did not result in asignificant reduction in tumor growth in this experiment. However,treatment with 18R5 and gemcitabine resulted in a reduction of tumorgrowth by 42% over treatment with gemcitabine (FIG. 12; p=<0.001 vsgemcitabine alone). Thus, 18R5 demonstrated synergistic anti-tumorgrowth activity in OMP-Pn4 pancreatic tumor model in combination withgemcitabine, an approved standard-of-care chemotherapy agent.

Reduction of PE-13 Breast Tumor Growth by Combination Treatment of 18R5and Paclitaxel

10.000 PE-13 human breast tumor cells (HER2-negative) were implanted inNOD-SCID mice and allowed to grow for 22 days until they reached anaverage volume of approximately 120 mm³. The animals were thenrandomized into 4 groups of 10 animals each and dosed with either acontrol antibody, anti-FZD 18R5, paclitaxel (TAXOL®®), or 18R5 pluspaclitaxel. FIG. 37 shows the average tumor volumes of the mice dosedwith control antibody, anti-FZD 18R5, paclitaxel, or 18R5 pluspaclitaxel. Antibodies were dosed at 10 mg/kg, IP, twice per week.Paclitaxel was dosed at 10 mg/kg, IP, once per week. Tumors weremeasured on the days indicated in FIG. 37. FIG. 38 shows the tumorgrowth of the individual animals in the 18R5 plus paclitaxel group. 18R5plus paclitaxel treatment was shown to result in anti-tumor activity andregression of established breast tumors.

Example 8 Assays to Determine Effect on Cancer Stem Cell Frequency

Limiting dilution assays (LDAs) can be used to assess the effect of aFZD-binding agent or antibody on solid tumor cancer stem cells and onthe tumorigenicity of a tumor comprising the cancer stem cells. Theassays can be used to determine the frequency of cancer stem cells intumors from animals treated with the FZD-binding antibody or other agentand to compare that frequency to the frequency of cancer stem cells intumors from control animals.

Effect of Combination Treatment of 18R5 and Irinotecan on Cancer StemCells in OMP-C28 Tumors

Control and treated tumors from the OMP-C28 xenograft study describedabove (Example 7) were harvested at the end of the study (day 48). Thetumors were processed and dissociated into single cells. Tumor cellswere then incubated with biotinylated mouse antibodies (α-mouseCD45-biotin 1:200 dilution and rat α-mouse H2 Kd-biotin 1:100 dilution,BioLegend, San Diego, Calif.) on ice for 30 min followed by addition ofstreptavidin-labeled magnetic beads (Invitrogen, Carlsbad, Calif.) toremove mouse cells with the aid of a magnet.

For the LDA, the human cells in the suspension were harvested, counted,and appropriate cell doses (5, 25, and 125 cells) in FACS buffer weremixed in a 1:1 mixture with Matrigel and injected subcutaneously inNOD/SCID mice (10 mice per cell dose per treatment group). Tumors areallowed to grow for up to 4 months. At the desired time point, thepercentage of mice with detectable tumors is determined in all groupsinjected with anti-FZD antibody treated tumor cells and compared to thepercentage of mice with detectable tumors in the controls. For example,the number of mice injected with 125 control-treated tumor cells thathave detectable tumors is determined and compared to the number of miceinjected with 125 FZD-antibody treated tumor cells that have detectabletumors. The cancer stem cell frequency is then calculated using L-Calc™software (StemCell Technologies Inc.). Briefly, based on Poissonstatistics, exactly one cancer stem cell exists among the known numberof injected cells if 37% of the animals fail to develop tumors.

For analysis of cell surface markers, the single tumor cell suspensionwas stained with anti-ESA (Biomeda) and anti-CD44 (BD Biosciences)antibodies which were directly conjugated to fluorochromes. Dead cellswere excluded by using the viability dye DAPI. Flow cytometry wasperformed using a FACS Aria (Becton Dickinson). Side scatter and forwardscatter profiles were used to eliminate cell clumps. Analysis of thetumors treated with control antibody revealed that 64% of the bulk tumorpopulation expressed both ESA and CD44 at high levels (FIG. 39). Thedouble positive population was not significantly affected by treatmentwith irinotecan alone (55%), as shown in FIG. 39, but treatment witheither 18R5 or the combination of 18R5 with irinotecan reduced thedouble positive population (40% and 32% respectively).

Effect of Combination Treatment of 18RS and Gemcitabine on Cancer StemCells in OMP-Pn4 Tumors

Control and treated tumors from the OMP-Pn4 xenograft study describedabove (Example 7) were harvested at the end of 41 days treatment. Thetumors were processed and dissociated into single cells. Tumor cellswere then incubated with biotinylated mouse antibodies (α-mouseCD45-biotin 1:200 dilution and rat α-mouse H2 Kd-biotin 1:100 dilution,BioLegend, San Diego, Calif.) on ice for 30 min followed by addition ofstreptavidin-labeled magnetic beads (Invitrogen, Carlsbad, Calif.) toremove mouse cells. The remaining human cells in the suspension werecollected, counted and diluted to appropriate cell doses (30, 90, 270and 810 cells), mixed in the mixture of 1:1 (v/v) FACS buffer andMatrigel and injected subcutaneously in NOD/SCID mice (10 mice per celldose per treatment group). Tumors were allowed to grow for 75 days asshown in FIG. 40. Each dot in FIG. 40 represents the tumor volume of anindividual mouse. The percentage of mice with detectable tumors wasdetermined in all groups injected with anti-FZD antibody treated tumorcells and compared to the percentage of mice with detectable tumors inthe controls. For example, the number of mice injected with 810control-treated tumor cells that have detectable tumors was determinedand compared to the number of mice injected with 810 FZD-antibodytreated tumor cells that have detectable tumors. The tumor growthfrequency was used to calculate the cancer stem cell frequency usingL-Calc™ software. The calculated cancer stem cell frequencies for eachof the treatment groups are shown in FIG. 41. Treatment with 18R5 aloneand treatment with 18R5 in combination with gemcitabine reduced cancerstem cell frequency, while treatment with gemcitabine alone had noeffect.

Example 9 Production of FZD antibodies

Antigen Production

Recombinant polypeptide fragments of the extracellular domain (ECD) orFri domain (Fri) of human FZD receptors (FZDs) are generated as antigensfor antibody production. Standard recombinant DNA technology is used toisolate polynucleotides encoding the amino acids of these domains of thedesired human frizzled receptor or receptors. These polynucleotides areligated in-frame N-terminal to either a human Fc-tag or histidine-tagand cloned into a transfer plasmid vector for baculovirus mediatedexpression in insect cells. Standard transfection, infection, and cellculture protocols are used to produce recombinant insect cellsexpressing the corresponding FZD polypeptides (O'Reilley et al.Baculovirus expression vectors: A Laboratory Manual, Oxford: OxfordUniversity Press (1994)).

Antigen protein is purified from insect cell conditioned medium usingProtein A and Ni++-chelate affinity chromatography. Purified antigenprotein is dialyzed against PBS (pH=7), concentrated to approximately 1mg/ml, and sterile filtered in preparation for immunization.

Immunization

Mice are immunized with purified FZD antigen protein using standardtechniques. Blood from individual mice are screened approximately 70days after initial immunization for antigen recognition using ELISA andFACS analysis (described in detail below). The two animals with thehighest antibody titers are selected for final antigen boost after whichspleen cells are isolated for hybridoma production. Hybridoma cells areplated at 1 cell per well in 96 well plates, and the supernatant fromeach well are screened by ELISA and FACS analysis against antigenprotein. Several hybridomas with high antibody titer are selected andscaled up in static flask culture. Antibodies are purified from thehybridoma supernatant using protein A or protein G agarosechromatography. Purified monoclonal antibodies are again tested by FACSand are isotyped to select for IgG and IgM antibodies.

Epilope Mapping

To identify antibodies that recognize specific regions of the FZDextracellular domain including the cysteine-rich domain, epitope mappingis performed. Mammalian expression plasmid vectors comprising a CMVpromoter upstream of polynucleotides that encode fragments of theextracellular FZD domain are generated using standard recombinant DNAtechnology. Recombinant proteins are then expressed in culturedmammalian cells by transient transfection. Twenty-four to 48 hoursfollowing transfection, cells are harvested and cell lysate proteinseparated on SDS-PAGE acrylamide gels for Western blotting usingantibodies from mice immunized with FZD antigen. Antibodies thatrecognize the ligand binding domain of FZD can be further analyzed forcompetitive binding with Wnt proteins by ELISA.

To identify specific epitopes within the extracellular domainsrecognized by an antibody against FZD the SPOTs system is used (SigmaGenosys, The Woodlands, Tex.). A series of 10-residue linear peptidesoverlapping by one amino acid and covering the entire FZD extracellulardomain are synthesized and covalently bound to a cellulose membrane bythe SPOT synthesis technique. The membrane is preincubated for 8 hoursat room temperature with blocking buffer and hybridized with antibodyovernight at 4° C. The membrane is then washed, incubated with asecondary antibody conjugated to horseradish peroxidase (HRP) (AmershamBioscience. Piscataway. NJ), re-washed, and visualized with signaldevelopment solution containing 3-amino-9-ethylcarbazole. Specificepitopes recognized by an antibody are thus determined.

FACS Analysis

To select monoclonal antibodies produced by hybridomas clones thatrecognize native cell-surface FZD protein. FACs analysis is used. HEK293cells are transfected with an expression vector encoding a full-lengthcDNA clone of the corresponding FZD either alone or co-transfected witha vector expressing GFP. A Flag epitope tag may be introduced at theamino-terminus, which allows verification of expression of the taggedFZD receptors on the cell surface. Twenty-four to 48-hourspost-transfection, cells are collected in suspension and incubated onice with anti-FZD antibodies, FLAG antibodies, immune serum (for FZD5expressing cells), or control IgG to detect background antibody binding.The cells are washed and primary antibodies detected with anti-mousesecondary antibodies conjugated to a fluorescent chromophore. Labeledcells are then sorted by FACS to identify anti-FZD antibodies thatspecifically recognize cell surface expression of the corresponding FZDreceptor. Antibodies that recognize the desired human frizzledreceptor(s) are identified.

Chimeric Antibodies

After monoclonal antibodies that specifically recognize a FZD receptorare identified, these antibodies are modified to overcome the humananti-mouse antibody (HAMA) immune response when rodent antibodies areused as therapeutics agents. The variable regions of the heavy-chain andlight-chain of the selected monoclonal antibody are isolated by RT-PCRfrom hybridoma cells and ligated in-frame to human IgG 1 heavy-chain andkappa light chain constant regions, respectively, in mammalianexpression vectors. Alternatively a human Ig expression vector such asTCAE 5.3 is used that contains the human IgG1 heavy-chain and kappalight-chain constant region genes on the same plasmid (Preston et al.,1998, Infection & Immunity 66:4137-42). Expression vectors encodingchimeric heavy- and light-chains are then co-transfected into Chinesehamster ovary (CHO) cells for chimeric antibody production.Immunoreactivity and affinity of chimeric antibodies are compared toparental murine antibodies by ELISA and FACS.

Humanized Antibodies

As chimeric antibody therapeutics are still frequently antigenic,producing a human anti-chimeric antibody (HACA) immune response,chimeric antibodies against a FZD receptor can undergo furtherhumanization. To generate humanized antibodies, key aspects of thespecificity determining motifs of the antibody, potentially includingelements from both the three short hypervariable sequences, orcomplementary determining regions (CDRs), and/or the framework regionsrequired to correctly position the CDR regions of the antibody heavy-and light-chain variable domains described above are engineered usingrecombinant DNA technology into the germline DNA sequences of humanheavy- and light-chain antibody genes, respectively, and then clonedinto a mammalian expression vector for expression in CHO cells. Theimmunoreactivity and affinity of the humanized antibodies are comparedto parental chimeric antibodies by ELISA and FACS. Additionally,site-directed or high-density mutagenesis of the variable region can beused to optimize specificity, affinity, etc. of the humanized antibody.

Human Antibodies

In some embodiments, human antibodies that specifically recognize theextracellular domain of a FZD receptor are isolated using phage displaytechnology. A phage display antibody library containing human antibodyvariable domains displayed as single chain Fv or as fab domains isscreened for specific and high affinity recognition of a FZD receptorantigen described above. The identified variable domain antibodysequences are then reformatted into an Ig expression vector containinghuman IgG1 heavy-chain and kappa light-chain for expression of humanantibodies in CHO cells.

Example 10 Production of Antibodies that Recognize Specific Epitopes

Monoclonal Antibodies from Hybridomas

In certain embodiments, antibodies recognizing functional epitopes ofFZD receptors are generated by immunizing mice with one or more of theFZD receptor antigens. Mice are immunized with the purified FZD antigenprotein using standard techniques. In certain embodiments mice areimmunized sequentially with distinct FZD receptor antigens. Blood fromindividual mice are screened approximately 70 days after initialimmunization. Animals with the high antibody titer for the FZD antigenare selected for final antigen boost after which spleen cells areisolated for hybridoma production. Hybridoma cells are plated at 1 cellper well in 96 well plates, and the supernatants from each well arescreened by ELISA and flow cytometry analysis. To identify monoclonalantibodies that recognize specific epitopes, including epitopes withinor overlapping with the Biological Binding Site (BBS), the hybridomasupernatant is screened both for antibody binding to the desired FZD(s)and for failure to bind to FZD that have specific amino acidsubstitutions within the desired specific epitope (e.g. the BBS).

Human Antibodies

A phage display library may be used to identify antibodies thatrecognize the desired epitopes of the FZD receptors (e.g., epitopescommon to multiple FZD and/or epitopes within or overlapping with theBBS or a portion thereof). For example, the Fri domain of a selected FZDis expressed as recombinant protein and coated on an appropriate surfaceat 10 μg/mL. A human phage library is then panned through two or morerounds of enrichment (See e.g. Griffiths et al., EMBO J. 12:715-34).Optionally, the subsequent rounds of panning may be performed usingdistinct FZD proteins. Optionally, each round of the panning may beperformed in the presence of decoy soluble FZD protein containingspecific amino acid substitutions within the desired target epitoperegion (e.g. including the epitopes within the Biological Binding Site(BBS)). Individual clones of the output from the panning selections arethen screened for the ability to bind to desired FZD protein(s) by ELISAor flow cytometry analysis and binding to a desired epitope is assessedby lack of binding to FZD protein containing specific amino acidsubstitutions within the desired target epitope. Genes encoding theantigen binding domain are then recovered from the phage and used toconstruct a complete human antibody molecule by joining the antigenbinding domain with constant regions for expression in a suitable hostcell line. Antibodies are identified and tested for the ability toprevent tumor cell growth as described elsewhere herein.

Example 11 Additional in vitro assays to evaluate antibodies against aFZD receptor

This example describes representative in vitro assays to test theactivity of antibodies generated against a FZD receptor on cellproliferation, pathway activation, and cytotoxicity.

Proliferation Assay

The expression of a FZD receptor by different cancer cell lines isquantified using Taqman analysis. Cell lines identified as expressing aFZD receptor are plated at a density of 104 cell per well in 96-welltissue culture microplates and allowed to spread for 24 hours.Subsequently cells are cultured for an additional 12 hours in fresh DMEMwith 2% FCS at which point anti-FZD antibodies versus control antibodiesare added to the culture medium in the presence of 10 μmol/L BrdU.Following BrdU labeling, the culture media is removed, and the cellsfixed at room temperature for 30 minutes in ethanol and reacted for 90minutes with peroxidase-conjugated monoclonal anti-BrdU antibody (cloneBMG 6H8, Fab fragments). The substrate is developed in a solutioncontaining tetramethylbenzidine and stopped after 15 minutes with 25 μlof 1 mol/L H₂SO₄. The color reaction is measured with an automatic ELISAplate reader using a 450 nm filter (UV Microplate Reader; Bio-RadLaboratories, Richmond, Calif.). All experiments are performed intriplicate. The ability of anti-FZD antibodies to inhibit cellproliferation compared to control antibodies is determined.

Pathway Activation Assay

In certain embodiments, the ability of antibodies against a FZD receptorto block activation of the Wnt signaling pathway is determined in vitro.For example, HEK 293 cells cultured in DMEM supplemented withantibiotics and 10% FCS are co-transfected with 1) Wnt7B and FZD10expression vectors to activate the Wnt signaling pathway; 2) a TCF/Lucwild-type or mutant reporter vector containing three or eight copies ofthe TCF-binding domain upstream of a firefly luciferase reporter gene tomeasure canonical Wnt signaling levels (Gazit et al., 1999, Oncogene18:5959-66): and 3) a Renilla luciferase reporter (Promega; Madison,Wis.) as an internal control for transfection efficiency. Anti-FZD10 andcontrol antibodies are then added to the cell culture medium.Forty-eight hours following transfection, luciferase levels are measuredusing a dual luciferase assay kit (Promega; Madison, Wis.) with fireflyluciferase activity normalized to Renilla luciferase activity. Threeindependent experiments are preformed in triplicate. The ability of theFZD antibodies to inhibit Wnt pathway activation is thus determined.

Complement-Dependent Cytotoxicity Assay

In certain embodiments, cancer cell lines expressing a FZD receptor orcancer stem cells isolated from a patient sample passaged as a xenograftin immunocompromised mice are used to measure complement dependentcytotoxicity (CDC) mediated by an antibody against a FZD receptor. Cellsare suspended in 200 μl RPMI 1640 culture medium supplemented withantibiotics and 5% FBS at 106 cells/ml. Suspended cells are then mixedwith 200 μl serum or heat-inactivated serum with antibodies against aFZD receptor or control antibodies in triplicate. Cell mixtures areincubated for 1 to 4 hours at 37° C. in 5% CO₂. Treated cells are thencollected, resuspended in 100 μl FITC-labeled annexin V diluted inculture medium and incubated at room temperature for 10 minutes. Onehundred microliters of a propidium iodide solution (25 μg/ml) diluted inHBSS is added and incubated for 5 minutes at room temperature. Cells arecollected, resuspended in culture medium and analyzed by flow cytometry.Flow cytometry of FITC stained cells provides total cell counts, andpropidium iodide uptake by dead cells as a percentage of total cellnumbers is used to measure cell death in the presence of serum andantibodies against a FZD compared to heat-inactivated serum and controlantibodies. The ability of anti-FZD antibodies to mediatecomplement-dependent cytotoxicity is thus determined.

Antibody-Dependent Cellular Cytotoxicity Assay

Cancer cell lines expressing a FZD receptor or cancer stem cellsisolated from a patient's sample passaged as a xenograft inimmunocompromised mice may be used to measure antibody dependentcellular cytotoxicity (ADCC) mediated by an antibody against a FZDreceptor. Cells are suspended in 200 μl phenol red-free RPMI 1640culture medium supplemented with antibiotics and 5% FBS at 10⁶ cells/ml.Peripheral blood mononuclear cells (PBMCs) are isolated from heparinizedperipheral blood by Ficoll-Paque density gradient centrifugation for useas effector cells. Target cells (T) are then mixed with PBMC effectorcells (E) at E/T ratios of 25:1, 10:1, and 5:1 in 96-well plates in thepresence of at least one FZD receptor antibody or a control antibody.Controls include incubation of target cells alone and effector cellsalone in the presence of antibody. Cell mixtures are incubated for 1 to6 hours at 37° C. in 5% CO2. Released lactate dehydrogenase (LDH), astable cytosolic enzyme released upon cell lysis, is then measured by acolorimetric assay (CytoTox96 Non-radioactive Cytotoxicity Assay;Promega: Madison, Wis.). Absorbance data at 490 nm are collected with astandard 96-well plate reader and background corrected. The percentageof specific cytotoxicity is calculated according to the formula: %cytotoxicity=100×(experimental LDH release−effector spontaneous LDHrelease−target spontaneous LDH release)/(target maximal LDHrelease−target spontaneous LDH release). The ability of antibodiesagainst a FZD receptor to mediate antibody dependent cellularcytotoxicity is thus determined.

Example 12 In Vivo Prevention of Tumor Growth Using Anti-FZD ReceptorAntibodies

This example describes a use of anti-FZD receptor antibodies to preventtumor growth in a xenograft model. In certain embodiments, tumor cellsfrom a patient sample (solid tumor biopsy or pleural effusion) that havebeen passaged as a xenograft in mice are prepared for repassaging intoexperimental animals. Tumor tissue is removed under sterile conditions,cut up into small pieces, minced completely using sterile blades, andsingle cell suspensions obtained by enzymatic digestion and mechanicaldisruption. Specifically, pleural effusion cells or the resulting tumorpieces are mixed with ultra-pure collagenase III in culture medium(200-250 units of collagenase per mL) and incubated at 37° C. for 3-4hours with pipetting up and down through a 10-mL pipette every 15-20minutes. Digested cells are filtered through a 45 pM nylon mesh, washedwith RPMI/20% FBS, and washed twice with HBSS. Dissociated tumor cellsare then injected subcutaneously into the mammary fat pads of NOD/SCIDmice to elicit tumor growth.

In certain embodiments, dissociated tumor cells are first sorted intotumorigenic and non-tumorigenic cells based on cell surface markersbefore injection into experimental animals. Specifically, tumor cellsdissociated as described above are washed twice with Hepes bufferedsaline solution (HBSS) containing 2% heat-inactivated calf serum (HICS)and resuspended at 106 cells per 100 μl. Antibodies are added and thecells incubated for 20 minutes on ice followed by two washes withHBSS/2% HICS. Antibodies include anti-ESA (Biomeda, Foster City,Calif.), anti-CD44, anti-CD24, and Lineage markers anti-CD2, —CD3.—CD10,—CD16, —CD18, —CD31, —CD64, and —CD140b (collectively referred to asLin; PharMingen, San Jose, Calif.). Antibodies are directly conjugatedto fluorochromes to positively or negatively select cells expressingthese markers. Mouse cells are eliminated by selecting against H2Kd+cells, and dead cells are eliminated by using the viability dye 7AAD.Flow cytometry is performed on a FACSVantage (Becton Dickinson, FranklinLakes, N.J.). Side scatter and forward scatter profiles are used toeliminate cell clumps. Isolated ESA+, CD44+, CD24−/low, Lin−tumorigeniccells are then injected subcutaneously into NOD/SCID mice to elicittumor growth.

By way of example, anti-FZD antibodies are analyzed for their ability toreduce the growth of tumor cells. Dissociated tumor cells (10,000 peranimal) are injected subcutaneously into the flank region of 6-8 weekold NOD/SCID mice. Two days after tumor cell injection, animals areinjected intraperitoneal (i.p.) with 10 mg/kg either anti-FZD antibodiestwo times per week. Tumor growth is monitored weekly until growth isdetected, after which point tumor growth is measured twice weekly for atotal of 8 weeks. FZD-binding antibodies which significantly reducetumor growth as compared to PBS injected controls are thus identified.

Example 13 In Vivo Treatment of Tumors Using Anti-FZD ReceptorAntibodies

This example describes a use of anti-FZD receptor antibodies to treatcancer in a xenograft model. In certain embodiments, tumor cells from apatient sample (solid tumor biopsy or pleural effusion) that have beenpassaged as a xenograft in mice are prepared for repassaging intoexperimental animals. Tumor tissue is removed, cut up into small pieces,minced completely using sterile blades, and single cell suspensionsobtained by enzymatic digestion and mechanical disruption. Dissociatedtumor cells are then injected subcutaneously either into the mammary fatpads, for breast tumors, or into the flank, for non-breast tumors, ofNOD/SCID mice to elicit tumor growth. Alternatively, ESA+, CD44+,CD24−/low, Lin−tumorigenic tumor cells are isolated as described aboveand injected.

Following tumor cell injection, animals are monitored for tumor growth.Once tumors reach an average size of approximately 150 to 200 mm,antibody treatment begins. Each animal receives 100 μg FZD receptorantibodies or control antibodies i.p. two to five times per week for atotal of 6 weeks. Tumor size is assessed twice a week during these 6weeks. The ability of FZD receptor antibodies to prevent further tumorgrowth or to reduce tumor size compared to control antibodies is thusdetermined.

At the end point of antibody treatment, tumors are harvested for furtheranalysis. In some embodiments a portion of the tumor is analyzed byimmunofluorescence to assess antibody penetration into the tumor andtumor response. A portion of each harvested tumor from anti-FZD receptortreated and control antibody treated mice is fresh-frozen in liquidnitrogen, embedded in O.C.T, and cut on a cryostat as 10 μm sectionsonto glass slides. In some embodiments, a portion of each tumor isformalin-fixed, paraffin-embedded, and cut on a microtome as 10 μmsection onto glass slides. Sections are post-fixed and incubated withchromophore labeled antibodies that specifically recognize injectedantibodies to detect anti-FZD receptor or control antibodies present inthe tumor biopsy. Furthermore antibodies that detect different tumor andtumor-recruited cell types such as, for example, anti-VE cadherin(CD144) or anti-PECAM-1 (CD31) antibodies to detect vascular endothelialcells, anti-smooth muscle alpha-actin antibodies to detect vascularsmooth muscle cells, anti-Ki67 antibodies to detect proliferating cells,TUNEL assays to detect dying cells, anti-β-catenin antibodies to detectWnt signaling, and anti-intracellular domain (ICD) Notch fragmentantibodies to detect Notch signaling can be used to assess the effectsof antibody treatment on, for example, angiogenesis, tumor growth andtumor morphology.

In certain embodiments, the effect of anti-FZD receptor antibodytreatment on tumor cell gene expression is also assessed. Total RNA isextracted from a portion of each harvested tumor from FZD antibodytreated and control antibody treated mice and used for quantitativeRT-PCR. Expression levels of FZD receptors, components of Wnt signalingpathway including, for example, Wnt1 and β-catenin, as well as additioncancer stem cell markers previously identified (e.g. CD44) are analyzedrelative to the house-keeping gene GAPDH as an internal control. Changesin tumor cell gene expression upon FZD receptor antibody treatment arethus determined.

In addition, the effect of anti-FZD receptor antibody treatment on thefrequency of cancer stem cells in a tumor is assessed. Tumor samplesfrom FZD versus control antibody treated mice are cut up into smallpieces, minced completely using sterile blades, and single cellsuspensions obtained by enzymatic digestion and mechanical disruption.Dissociated tumor cells are then analyzed by FACS analysis for thepresence of tumorigenic cancer stem cells based on ESA+, CD44+.CD24−/low, Lin−surface cell marker expression as described in detailabove.

The tumorigenicity of cells isolated based on ESA+, CD44+, CD24−/low,Lin− expression following anti-FZD antibody treatment can then assessed.ESA+, CD44+, CD24−/low, Lin− cancer stem cells isolated from FZDantibody treated versus control antibody treated mice are re-injectedsubcutaneously into the mammary fat pads of NOD/SCID mice. Thetumorigenicity of cancer stem cells based on the number of injectedcells required for consistent tumor formation is then determined.

Example 14 Identification of a Wnt Gene Signature

Experiments were conducted to identify a group of genes whose expressionis specific for Wnt signaling pathway activation in human colon tumors.

Abrogation of Tumor Growth by Axin Overexpression

Axin is an important regulator of the canonical Wnt pathway. It is partof the multiprotein complex that triggers β-catenin degradation, thuskeeping the pathway silent in the absence of Wnt. This effect isreversed by Wnt, which removes axin from the destruction complex,allowing for β-catenin translocation and TCF-mediated activation ofspecific target genes. Both exogenous axin over-expression andexpression of a dominant negative truncated form of TCF (DNTCF4)represent well-characterized means to block the Wnt signaling pathway.

We showed that lentivirus-mediated axin overexpression completelyabrogated the growth of UM-PE13 and UM-T3 breast tumors as well as thegrowth of OMP-C11 and OMP-C17 colon tumors in NOD/SCID mice. Stableexpression of DNTCF4 in UM-T3 tumor cells had the same effect. Takentogether, these data demonstrate that intracellular Wnt blockade cannegatively affect the development of different tumor types, supportingthe Wnt pathway as a relevant target for the treatment of breast andcolon cancers.

The Wnt signaling pathway is constitutively activated in many tumortypes. In most colon tumors this activation is due to truncatingmutation of APC or activating mutations of β-catenin. Such mutationshave not been reported for other tissues, in which the Wnt signalingpathway could be activated through another set of mutations or anautocrine mechanism. In those tumors where the Wnt signaling pathwayremains responsive to autocrine stimuli, blocking the pathway usingextracellular means such as antibodies or other soluble proteininhibitors should be feasible and impact tumor development. Identifyingsuch Wnt-dependent tumors would be helpful in developing anti-Wnt agentsand defining tumor types to target in the clinic.

Immunohistochemical data showed that most OMP-C11 tumor cells expresshigh levels of cytoplasmic/nuclear β-catenin, suggesting that the Wntsignaling pathway is constitutively activated in this tumor type. Thiswas confirmed by the detection of high levels of β-catenin in OMP-C11 byWestern blot. The combination of Wnt pathway activation and sensitivityto axin overexpression makes OMP-C11 a good tumor in which to study theregulation of gene expression in response to Wnt and Wnt blockade andfrom which to derive a Wnt gene signature.

Microarray Analysis of Differential Gene Expression in Response to AxinOverexpression

The differential gene expression upon treatment of OMP-C11 colon tumorcells with axin was determined by microarray analysis.

Human colon OMP-C11 tumors freshly removed from NOD/SCID mice (xenografttumor model) were used as a source for the colon tumor cells. Twolentiviral vectors were generated for the delivery of a constitutiveaxin-IRES-GFP expression cassette and a control IRES-GFP expressioncassette that were termed LOM91 and LOM92, respectively.

OMP-C11 tumors were processed to a single cell suspension and depletedfrom the mouse lineage cells. The lin-depleted cells were infected withLOM91 (axin) or 92 (control) lentiviral vectors using a multiplicity ofinfection of 2.5, maintained in culture for 3-4 days and sorted for GFPexpression. Total RNA was extracted from each sample of sorted cells.The RNAs were analyzed on the GeneChip® Human Genome U133 Plus 2.0microarray (Affymetrix, Santa Clara, Calif.). The experiment wasrepeated twice.

A gene signature containing a core set of genes regulated by the Wntpathway was generated by analysis of the genes that are differentiallyexpressed following axin treatment and that also exhibit correlationwith the expression of axin2 across a panel of normal and malignantcolon tumor samples. Genes were identified from the axin microarrayexperiment (above) that showed down regulation in response to axisoverexpression. The cutoff for this selection was down-regulation by 50%or more in Axin1 over-expressing samples comparing to control samples(log 2ratio of Axin1 over-expressing samples over control samples has tobe −1 or smaller), with T test p value smaller than 0.1. As Axin1 is aknown Wnt pathway inhibitor, genes down-regulated by Axin1 overexpression will be direct or indirect Wnt pathway targets. Thisselection was then further refined by identification of those geneswhich showed high correlation (correlation value >0.3) with axin2 amonga set of colon/intestine/other digestive tissue malignant tumor samples(232 samples). Since Axin2 is a known Wnt target, genes showing similarexpression pattern as Axin2 will likely be Wnt target as well. Thisanalysis produced a gene signature for Wnt pathway activity (Table 6).The expression levels of the genes in this signature can be used toassess whether individual tumor samples or different types of tumorsshow evidence of altered Wnt pathway signaling.

TABLE 6 Wnt gene signature list derived from colon tumors HG_U133_Plus_2Gene Probe Set ID Correlation log2ratio Symbol Description 222696_at 1−1.52705 AXIN2 axin 2 (conductin, axil) 206286_s_at 0.81941 −1.15086TDGF1 teratocarcinoma-derived growth factor 1 213880_at 0.787599−3.32482 LGR5 leucine-rich repeat-containing G protein-coupled receptor5 207217_s_at 0.781543 −1.15534 NOX1 NADPH oxidase 1 209588_at 0.762464−1.09556 EPHB2 EPH receptor B2 212850_s_at 0.75362 −2.00386 LRP4 lowdensity lipoprotein receptor-related protein 4 205107_s_at 0.744062−1.01296 EFNA4 ephrin-A4 214058_at 0.718735 −1.74381 MYCL1 v-mycmyelocytomatosis viral oncogene homolog 1, lung carcinoma derived(avian) 209864_at 0.714811 −1.25716 FRAT2 frequently rearranged inadvanced T-cell lymphomas 2 208121_s_at 0.711694 −1.87702 PTPRO proteintyrosine phosphatase, receptor type, O 229376_at 0.711672 −1.73296Unknown expressed sequence tag (EST) 202431_s_at 0.659255 −1.49975 MYCv-myc myelocytomatosis viral oncogene homolog (avian) 212444_at 0.656023−2.15908 GPCR5A G protein-coupled receptor, family C, group 5, member A222938_x_ at 0.654955 −2.43139 ENPP3 ectonucleotide pyrophosphatase/phosphodiesterase 3 241607_at 0.640554 −1.00413 LOC730102 hypotheticalprotein LOC730102 227475_at 0.637963 −3.60987 FOXQ1 forkhead box Q1230398_at 0.628654 −1.65124 TNS4 tensin 4 40284_at 0.601382 −1.21862FOXA2 forkhead box A2 219704_at 0.557276 −1.33216 YBX2 Y box bindingprotein 2

Example 15 Treatment of Human Cancer Using Anti-FZD Receptor Antibodies

This example describes methods for treating cancer using antibodiesagainst a FZD receptor to target tumors comprising cancer stem cellsand/or tumor cells in which FZD receptor expression has been detectedand/or tumor cells having a Wnt gene signature indicating that they areresponsive to inhibition of Wnt signaling (e.g., the Wnt gene signatureof Example 14).

The presence of cancer stem cell marker or FZD receptor or theexpression of one or more genes in a Wnt gene signature can first bedetermined from a tumor biopsy. Tumor cells from a biopsy from a patientdiagnosed with cancer are removed under sterile conditions. In someembodiments the tissue biopsy is fresh-frozen in liquid nitrogen,embedded in O.C.T., and cut on a cryostat as 10 μm sections onto glassslides. In some embodiments, the tissue biopsy is formalin-fixed,paraffin-embedded, and cut on a microtome as 10 μm section onto glassslides.

Sections are incubated with antibodies against a FZD receptor to detectFZD protein expression. Alternatively, sections can be analyzed for thepresence of one or more genes in the Wnt gene signature as described inExample 14.

The presence of cancer stem cells also may be determined. Tissue biopsysamples are cut up into small pieces, minced completely using sterileblades, and cells subject to enzymatic digestion and mechanicaldisruption to obtain a single cell suspension. Dissociated tumor cellsare then incubated with anti-ESA, -CD44, -CD24, -Lin, and -FZDantibodies to detect cancer stem cells, and the presence of ESA+. CD44+,CD24−/low, Lin−, FZD+ tumor stem cells is determined by flow cytometryas described in detail above.

Cancer patients whose tumors are diagnosed as expressing a FZD receptorand/or one or more genes in the Wnt gene signature are treated withanti-FZD receptor antibodies. In certain embodiments, humanized or humanmonoclonal anti-FZD receptor antibodies generated as described above arepurified and formulated with a suitable pharmaceutical vehicle forinjection. In some embodiments, patients are treated with the FZDantibodies at least once a month for at least 10 weeks. In someembodiments, patients are treated with the FZD antibodies at least oncea week for at least about 14 weeks. Each administration of the antibodyshould be a pharmaceutically effective dose. In some embodiments,between about 2 to about 100 mg/ml of an anti-FZD antibody isadministered. In some embodiments, between about 5 to about 40 mg/ml ofan anti-FZD antibody is administered. The antibody can be administeredprior to, concurrently with, or after standard radiotherapy regimens orchemotherapy regimens using one or more chemotherapeutic agent, such asoxaliplatin, fluorouracil, leucovorin, or streptozocin. Patients aremonitored to determine whether such treatment has resulted in ananti-tumor response, for example, based on tumor regression, reductionin the incidences of new tumors, lower tumor antigen expression,decreased numbers of cancer stem cells, or other means of evaluatingdisease prognosis.

Example 16 Differentiation of Pancreatic Tumor Cells Following Treatmentwith 18R5 and Gemcitabine

Gene Expression Analysis of Treated Pancreatic Tumor Cells byQuantitative PCR (Q-PCR)

PN4 xenograft tumors treated with either control Ab, 18R5 IgG antibody,gemcitabine, or the combination of gemcitabine and 18R5. IgG antibody asdescribed above (Example 7) were analyzed for expression of chromograninA (CHGA) by quantitative PCR analysis. CHGA is well known to be a markerfor neuroendocrine differentiation of various tumors including breast,colon, lung and pancreatic tumors and elevated expression of CHGA inpancreatic tumors has been found to be associated with improved survival(Tezel et al. 2000. Cancer 89, 2230-6).

Total RNA was prepared from 5-tumors of each group in the PN4 xenograftstudy and was evaluated by one-step reverse transcription (RT)-PCR usingApplied Biosystems Taqman® inventoried probes according to standardprotocols. The probe-primer set (Hs00154441_ml 1) used for analysis ofCHGA included a FAM-dye labeled probe and the following primer:5′-CGCTCTCCAAGGCGCCAAGGAGAGG-3′ (SEQ ID NO:75). Gus B was used asinternal control. Briefly, RT was done at 48° C. for 30 min, initialdenaturation at 95° C. for 10 min, followed by 40 cycles of denaturationat 95° C. for 15 seconds, and extension at 60° C. for 1 min and theamplification/incorporation of fluorescent probes was observedreal-time.

The islet beta cell marker CHGA was elevated significantly only insamples from mice treated with both gemcitabine and 18R5. Tumors fromthe control Ab, 18R5, and gemcitibine alone groups expressed similarlevels of CHGA RNA while tumors from the combination group showed aclear increase in CHGA expression. CHGA levels were elevated 10-fold and7-fold in two experiments in tumors treated with both 18R5 andgemcitabine. The results of a representative experiment are shown inFIG. 42.

Gene Expression Analysis of Treated Pancreatic Tumor Cells byImmunohistochemistry

The increased expression of CHGA was also observed at the protein levelby immunohistochemistry on tissue sections prepared from treated tumors(data not shown). Control Ab treated tumors showed intense staining of asmall subset of cells scattered throughout tumors. Tumors treated with18R5 alone or gemcitabine alone expressed CHGA at similar levels as thecontrols. In contrast, tumors treated with the combination of 18R5 andgemcitabine showed an increase in the number of CHGA-positive cells,consistent with the increased RNA expression detected by Q-PCR.

Staining with Alcian Blue and Antibody to Ki67

Another characteristic of endocrine, secretory or ductal cells is theproduction of mucin and these cells can be detected by alcian bluestaining (van Es et al. 2005. Nature 435 959-63). It was observed duringharvesting and processing of tumors that the 18R5 treated tumors weremuch more mucinous than control treated tumors. Therefore, PN4 tumorssections from mice treated with control antibody, gemcitabine alone,18R5 alone, or 18R5 plus gemcitabine were stained with alcian blue. The18R5 treated tumors showed a clear increase in alcian blue staining inboth the 18R5 alone and the 18R5 plus gemcitabine groups relative tocontrols and the gemcitabine alone group (data not shown).

Increased mucinous cells were also noted in a second pancreatic tumorline, PN13, following treatment with 18R5 or control antibody (FIG. 43).In this experiment mice bearing PN13 tumors were treated as describedabove (Example 7). Tumors sections were stained with alcian blue toreveal mucinous cells and also stained by immunohistochemistry with anantibody to ki67 to reveal cells undergoing proliferation. The resultsshow that 18R5 treatment resulted in greatly increased numbers of alcianblue positive mucinous cells. Additionally, the frequency of ki67positive cells was reduced by 1 8R5 treatment. Interestingly there wasnot an overlap between the mucinious cells and cells that were ki67positive, suggesting that the mucinous cells are not proliferative. Thisprovides evidence that 18R5 treatment is promoting the differentiationof tumor cells into non-proliferative progeny.

In summary, the increased expression of CHGA, the increased productionof mucins as evidenced by alcian blue staining, and the production ofnon-proliferative progeny as evidenced by staining with antibody to ki67are consistent with a model that inhibiting Wnt-FZD signaling with 18R5treatment promotes the differentiation of pancreatic tumor cells towardsmultiple distinct cell types with features characteristic ofnon-proliferative differentiated cells.

Example 17 Additional In Vivo Efficacy Studies with 18R5 Alone and/or inCombination with Other Anti-Cancer Agents

Effect on OMP-L U24 Xenograft Tumor Growth

The efficacy of anti-FZD antibody 18R5, both alone and in combinationwith Taxol® (paclitaxel), in inhibiting the growth of OMP-LU24 humanlung tumors in vivo was assessed.

50,000 OMP-LU24 human lung tumor cells were injected subcutaneously inNOD-SCID mice. Tumors were allowed to grow for 27 days until theyreached an average volume of 143 mm³. The animals were randomized into 4groups (n=9 per group) and treated with either control antibody(“Control Ab”), anti-FZD 18R5 (“18R5”). Taxol® (“Taxol”) or thecombination of 18R5 plus Taxol® (“18R5+Taxol”). Tumor measurements weremade on the days indicated in FIG. 44. Antibodies were dosed at 10 mg/kgintraperitoneal (IP), once per week and Taxol® was dosed at 15 mg/kg,IP, once per week.

The results are shown in FIG. 44. Anti-FZD treatment was seen to reducetumor growth, and the combination treatment showed enhanced anti-tumoractivity relative to Taxol®® alone.

Effect on OMP-LU33 Xenograft Tumor Growth

The efficacy of anti-FZD antibody 18R5, both alone and in combinationwith Avastin® (bevacizumab), in inhibiting the growth of OMP-LU33 humanlung tumors in vivo was also tested.

10,000 OMP-LU33 human lung tumor cells were injected subcutaneously inNOD-SCID mice. Tumors were allowed to grow for 30 days until theyreached an average volume of 124 mm³. The animals were randomized into 4groups (n=10 per group) and treated with either control antibody(squares), Avastin® (triangles pointing down), anti-FZD 18R5 (trianglespointing up), or the combination of 18R5 plus Avastin® (circles). Tumormeasurements were made on the days indicated in FIG. 45. Antibodies weredosed at 10 mg/kg IP, twice per week.

The results are shown in FIG. 45. Anti-FZD treatment was seen to reducetumor growth, and the combination treatment showed enhanced anti-tumoractivity relative to Avastin® alone.

Effect On T3 Xenograft Tumor Growth

The efficacy of anti-FZD antibody 18R5, both alone and in combinationwith Herceptin® (trastuzumab), in inhibiting the growth of T3 humanHER2-positive breast tumors in vivo was also assessed.

50,000 T3 human breast tumor cells were injected subcutaneously inNOD-SCID mice. Tumors were allowed to grow for 32 days until theyreached an average volume of 125 mm³. The animals were randomized into 4groups (n=10 per group) and treated with either control antibody(squares), anti-FZD 18R5 (triangles), Herceptin, (small filled circles),or the combination of 185 plus Herceptin® (open circles). Tumormeasurements were made on the days indicated in FIG. 46. Antibodies weredosed at 10 mg/kg IP, twice per week.

The results are shown in FIG. 46. The combination treatment with 18R5and Herceptin® showed enhanced anti-tumor activity relative toHerceptin,® alone.

Example 18 Anti-FZD Antibody Sequences

Heavy chain and light chain CDRs of anti-FZD antibodies are provided inTables 7 and 8, below, respectively. The heavy chain variable regions(VH) and light chain variable regions (VL) of the anti-FZD antibodiesand their coding sequences are identified in Table 9, below. The aminoacid and polynucleotide sequences of the VH and VL listed in Table 9 areprovided in FIGS. 13-15 or Tables 10 and 11, below. Sequences encodingthe heavy chain or light chains of the anti-FZD antibodies are providedin FIGS. 14-15 or Table 12, below.

TABLE 7 Heavy chain CDRs of anti-FZD human antibodies Heavy Chain Ab(s)CDR1 CDR2 CDR3 18R8 GFTFSHYTLS VISGDGSYTYYADSVKG NFIKYVFAN (SEQ ID(SEQ ID NO: 2) (SEQ ID NO: 1) NO: 3) 18R5 GFTFSHYTLS VISGDGSYTYYADSVKGNFIKYVFAN 18R4605 (SEQ ID (SEQ ID NO: 2) (SEQ ID 18R4805 NO: 1) NO: 3)44R24 GFTFSSYYIT TISYSSSNTYYADSVKG SIVFDY (SEQ ID (SEQ ID NO: 78)(SEQ ID NO: 77) NO: 79)

TABLE 8 Light chain CDRs of anti-FZD human antibodies Light Chain Ab(s)CDR1 CDR2 CDR3 18R8 SGDKLGKKYAS EKDNRPSG SSFAGNSLE (SEQ ID (SEQ ID(SEQ ID NO: 4) NO: 5) NO: 6) 18R5 SGDNIGSFYVH DKSNRPSG QSYANTLSL 18R4605(SEQ ID (SEQ ID (SEQ ID 18R4805 NO: 7) NO: 8) NO: 9) 44R24 SGDALGNRYVYSG GSWDTRPYPKY (SEQ ID (SEQ ID (SEQ ID NO: 80) NO: 81) NO: 82)

TABLE 9 VH and VL of ant-FZD human antibodies Heavy Chain Light ChainHeavy Chain Light Chain Variable Variable Variable Variable Region (VH)Region (VL) Region (VH) Region (VL) amino acid amino acid Coding CodingAb(s) sequence sequence Sequence Sequence 18R8 SEQ ID NO: 10 SEQ ID NO:12 SEQ ID NO: 17 SEQ ID NO: 19 18R8 SEQ ID NO: 10 SEQ ID NO: 12 SEQ IDNO: 87 SEQ ID NO: 88 (codon optimized) 18R5 SEQ ID NO: 10 SEQ ID NO: 14SEQ ID NO: 17 SEQ ID NO: 21 18R5 SEQ ID NO: 10 SEQ ID NO: 14 SEQ ID NO:87 SEQ ID NO: 89 (codon optimized) 18R4605 SEQ ID NO: 10 SEQ ID NO: 14SEQ ID NO: 87 SEQ ID NO: 90 18R4805 SEQ ID NO: 10 SEQ ID NO: 14 SEQ IDNO: 87 SEQ ID NO: 92 44R24 SEQ ID NO: 85 SEQ ID NO: 86 SEQ ID NO: 94 SEQID NO: 95

TABLE 10 Additional anti-FZD VH and VL amino acid sequencesSEQUENCE (SEQ ID NO:) 44R24 VH (SEQ ID NO: 85):EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYITWVRQAPGKGLEWVSTISYSSSNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSIVFDYWGQGTLVTVSS44R24 VL (SEQ ID NO: 86):DIELTQPPSVSVAPGQTARISCSGDALGNRYVYWYOQKPGQAPVLVIPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCGSWDTRPYPKYVFGGGTKLTVLG

TABLE 11 Additional nucleotide sequences encoding VH and VL ofanti-FZD antibodies SEQUENCE (SEQ ID NO:)18R5/18R8 VH coding sequence (codon-optimized) (SEQ ID NO: 87):GAGGTGCAGCTGGTCGAGTCTGGCGGCGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCCTGCGCCGCCTCCGGCTTCACCTTCTCCCACTACACCCTGTCCTGGGTGCGCCAGGCAGCCGACTCCGTGAAGGGCCGGTTCACCATCTCCTCCGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCTCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGCCCGGAACTTCATCAAGTACGTGTTCGCCAACTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC18R8 VL coding sequence (codon-optimized) (SEQ ID NO: 88):GACATCGAGCTGACCCAGCCTCCCTCCGTGTCTGTGGCTCCTGGCCAGACCGCCCGGATCTCCTGCTCCGGCGACAAGCTGGGCAAGAAGTACGCCTCCTGGTATCAGCAGAAGCCTGGACAGGCCCCTGTGCTGGTCATCTACGAGAAGGACAACCGGCCTAGCGGCATCCCTGAGCGGTTCTCCGGCTCCAACTCCGGCAACACCGCCACCCTGACCATCTCCGGCACCCAGGCCGAGGACGAGGCCGACTACTACTGCTCCTCCTTCGCCGGCAACTCCCTGGAAGTGTTCGGCGGAGGCACCAAGCTGACCGTGCTGGGC18R5 VL coding sequence (codon-optimized) (SEQ ID NO: 89):GACATCGAGCTGACCCAGCCTCCCTCCGTGTCCGTGGCCCCTGGCCAGACCGCCCGGATCTCCTGCTCCGGCGACAACATCGGCAGCTTCTACGTGCACTGGTATCAGCAGAAACCTGGACAGGCCCCTGTGCTGGTGATCTACGACAAGTCCAACCGGCCTTCCGGCATCCCTGAGCGGTTCTCCGGCTCCAACTCCGGCAACACCGCCACCCTGACCATCTCCGGCACCCAGGCCGAGGACGAGGCCGACTACTACTGCCAGTCCTACGCCAACACCCTGTCCCTGGTGTTTGGCGGCGGAACAAAGCTGACCGTGCTGGGC 18R4605 VL coding sequence (SEQ ID NO: 90):GACATAGAACTAACTCAGCCACCCTCTGTTAGCGTTGCACCGGGACAGACGGCACGTATATCGTGCTCGGGAGACAATATAGGAAGTTTCTATGTACATTGGTATCAACAGAAACCTGGTCAAGCACCTGTATTAGTAATCTATGACAAAAGTAACCGACCTTCCGGAATACCTGAGCGTTTCAGTGGTTCGAACTCCGGCAACACTGCAACTTTAACTATATCTGGAACTCAGGCGGAGGATGAGGCTGACTACTACTGCCAGAGTTACGCAAACACTCTGTCCCTGGTGTTTGGCGGCGGAACAAAGTTAACCGTGCTGGGC 18R4805 VL coding sequence (SEQ ID NO: 92):GACATAGAACTAACTCAGCCGCCGTCTGTTAGCGTTGCACCGGGACAGACGGCACGTATATCGTGCTCGGGAGACAATATTGGTTCTTTCTATGTACATTGGTATCAACAGAAACCTGGTCAAGCACCTGTATTAGTAATATATGACAAAAGTAACCGTCCTTCGGGAATACCTGAGCGTTTCAGTGGTTCGAACTCGGGCAACACTGCAACTTTAACTATATCTGGAACGCAGGCGGAGGATGAGGCGGACTACTATTGCCAAAGTTACGCAAACACTCTATCCTTAGTGTTTGGTGGAGGAACAAAGTTAACCGTGCTAGGC 44R24 VH coding sequence (SEQ ID NO: 94):GAGGTGCAGCTGGTGGAGTCTGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCTCTTACTACATCACCTGGGTGCGCCAGGCTCCTGGCAAGGGACTGGAATGGGTGTCCACCATCTCCTACTCCTCCAGCAACACCTACTACGCCGACTCCGTGAAGGGCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCCGGTCCATCGTGTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCT44R24 VL coding sequence (SEQ ID NO: 95):GACATCGAGCTGACCCAGCCTCCCTCTGTGTCTGTGGCCCCTGGCCAGACCGCCAGGATCTCTTGCTCTGGCGACGCCCTGGGCAACAGATACGTGTACTGGTATCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTGATCCCTTCCGGCATCCCTGAGCGGTTCTCCGGCTCCAACTCCGGCAACACCGCCACCCTGACCATCTCTGGCACCCAGGCCGAGGACGAGGCCGACTACTACTGCGGCTCCTGGGACACCCGGCCTTACCCTAAGTACGTGTTCGGCGGAGGCACCAAGCTGACCGTGCTGGGC

TABLE 12 Additional nucleotide sequences encoding heavy chains (HC) orlight chains (LC) of anti-FZD IgG antibodies (includingsignal sequences) SEQUENCE (SEQ ID NO:)18R5/18R8/18R4605/18R4805 IgG2 HC coding sequence(codon-optimized) (SEQ ID NO: 96):ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTCGCCGCTCCTAGATGGGTGCTGTCCGAGGTGCAGCTGGTCGAGTCTGGCGGCGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCCTGCGCCGCCTCCGGCTTCACCTTCTCCCACTACACCCTGTCCTGGGTGCGCCAGGCACCAGGGAAGGGACTGGAGTGGGTCTCCGTGATCTCCGGCGACGGCTCCTACACCTACTACGCCGACTCCGTGAAGGGCCGGTTCACCATCTCCTCCGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCTCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGCCCGGAACTTCATCAAGTACGTGTTCGCCAACTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCCGCCTCCACCAAGGGCCCTTCCGTGTTCCCTCTGGCCCCTTGCTCCCGGTCCACCTCCGAGTCCACCGCCGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACAGTGCCTTCCTCCAACTTCGGCACCCAGACCTACACCTGCAACGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGACCGTGGAGCGGAAGTGCTGCGTGGAGTGCCCTCCTTGCCCTGCCCCTCCTGTGGCTGGTCCTAGCGTGTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGGACCCCTGAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGATCCTGAAGTCCAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAGCAGTTCAACTCCACCTTCCGGGTGGTGTCCGTGCTGACCGTGGTGCACCAGGACTGGCTGAACGGCAAAGAATACAAGTGCAAAGTCTCCAACAAGGGCCTGCCTGCCCCTATCGAAAAGACCATCAGCAAGACCAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCTCCCTCTCGCGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAGTGGGAGTCCAACTTCCTGTACTCCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGTCCCTGTCCCTGTCT CCTGGCAAG18R8 lambda LC coding sequence (codon-optimized) (SEQ ID NO: 97):ATGGCCTGGGCCCTGCTGCTGCTGACCCTGCTGACACAGGGCACCGGCTCTTGGGCCGACATCGAGCTGACCCAGCCTCCCTCCGTGTCTGTGGCTCCTGGCCAGACCGCCCGGATCTCCTGCTCCGGCGACAAGCTGGGCAAGAAGTACGCCTCCTGGTATCAGCAGAAGCCTGGACAGGCCCCTGTGCTGGTCATCTACGAGAAGGACAACCGGCCTAGCGGCATCCCTGAGCGGTTCTCCGGCTCCAACTCCGGCAACACCGCCACCCTGACCATCTCCGGCACCCAGGCCGAGGACGAGGCCGACTACTACTGCTCCTCCTTCGCCGGCAACTCCCTGGAAGTGTTCGGCGGAGGCACCAAGCTGACCGTGCTGGGCCAGCCTAAGGCCGCTCCTTCCGTGACCCTGTTCCCTCCTTCCTCCGAGGAACTGCAGGCCAACAAGGCCACCCTGGTCTGCCTGATCTCCGACTTCTACCCTGGCGCCGTGACCGTGGCCTGGAAGGCCGACTCCTCCCCTGTGAAGGCCGGCGTGGAGACAACCACCCCTTCCAAGCAGTCCAACAACAAGTACGCCGCCTCCTCCTACCTGTCCCTGACCCCTGAGCAGTGGAAGTCCCACCGGTCCTACTCTTGCCAGGTCACCCACGAGGGCTCCACCGTGGAAAAGACAGTGGCCCCCACCGAGTGCTCC18R5 LC coding sequence (codon-optimized) (SEQ ID NO: 76):ATGGCCTGGGCCCTGCTGCTGCTGACCCTGCTGACACAGGGCACCGGCTCTTGGGCCGACATCGAGCTGACCCAGCCTCCCTCCGTGTCCGTGGCCCCTGGCCAGACCGCCCGGATCTCCTGCTCCGGCGACAACATCGGCAGCTTCTACGTGCACTGGTATCAGCAGAAACCTGGACAGGCCCCTGTGCTGGTGATCTACGACAAGTCCAACCGGCCTTCCGGCATCCCTGAGCGGTTCTCCGGCTCCAACTCCGGCAACACCGCCACCCTGACCATCTCCGGCACCCAGGCCGAGGACGAGGCCGACTACTACTGCCAGTCCTACGCCAACACCCTGTCCCTGGTGTTTGGCGGCGGAACAAAGCTGACCGTGCTGGGCCAGCCTAAGGCCGCTCCTTCCGTGACCCTGTTCCCTCCTTCCTCCGAGGAGCTGCAGGCCAACAAGGCCACCCTGGTGTGCCTGATCTCCGACTTCTACCCTGGCGCTGTGACTGTGGCTTGGAAGGCCGACTCCTCCCCTGTGAAGGCCGGCGTGGAGACAACCACCCCTTCCAAGCAGTCCAACAACAAGTACGCCGCCTCCTCCTACCTGTCCCTGACCCCTGAGCAGTGGAAGTCCCACCGGTCCTACTCTTGCCAGGTGACCCACGAGGGCTCCACCGTGGAAAAGACAGTGGCACCCACCGAGTGCTCC18R4605 LC coding sequence (SEQ ID NO: 83):ATGGCATGGGCATTATTGCTACTTACTCTATTGACGCAAGGAACGGGTTCATGGGCAGACATAGAACTAACTCAGCCACCCTCTGTTAGCGTTGCACCGGGACAGACGGCACGTATATCGTGCTCGGGAGACAATATAGGAAGTTTCTATGTACATTGGTATCAACAGAAACCTGGTCAAGCACCTGTATTAGTAATCTATGACAAAAGTAACCGACCTTCCGGAATACCTGAGCGTTTCAGTGGTTCGAACTCCGGCAACACTGCAACTTTAACTATATCTGGAACTCAGGCGGAGGATGAGGCTGACTACTACTGCCAGAGTTACGCAAACACTCTGTCCCTGGTGTTTGGCGGCGGAACAAAGTTAACCGTGCTGGGCCAGCCTAAGGCCGCACCTTCGGTGACCCTATTCCCTCCTTCATCCGAGGAGCTACAGGCCAACAAGGCCACCTTAGTGTGCCTAATCTCCGACTTCTATCCTGGTGCTGTAACGGTAGCGTGGAAGGCCGACTCATCGCCGGTGAAGGCCGGTGTGGAGACAACGACTCCTTCCAAGCAGTCCAACAACAAATACGCCGCGTCCTCCTACCTGTCCCTAACCCCTGAGCAGTGGAAGTCCCACCGTTCATACTCGTGCCAGGTGACGCACGAGGGTTCAACGGTCGAAAAGACAGTAGCACCTACTGAATGCTCA18R4805 LC coding sequence (SEQ ID NO: 84):ATGGCATGGGCATTATTACTACTTACTCTACTTACGCAAGGAACGGGTTCATGGGCAGACATAGAACTAACTCAGCCGCCGTCTGTTAGCGTTGCACCGGGACAGACGGCACGTATATCGTGCTCGGGAGACAATATTGGTTCTTTCTATGTACATTGGTATCAACAGAAACCTGGTCAAGCACCTGTATTAGTAATATATGACAAAAGTAACCGTCCTTCGGGAATACCTGAGCGTTTCAGTGGTTCGAACTCGGGCAACACTGCAACTTTAACTATATCTGGAACGCAGGCGGAGGATGAGGCGGACTACTATTGCCAAAGTTACGCAAACACTCTATCCTTAGTGTTTGGTGGAGGAACAAAGTTAACCGTGCTAGGCCAGCCTAAGGCCGCACCTTCGGTGACCCTATTCCCTCCTTCATCCGAGGAGCTACAGGCGAACAAAGCCACCTTAGTGTGCCTAATCTCAGACTTTTATCCTGGTGCTGTAACGGTAGCGTGGAAGGCGGACTCATCGCCGGTGAAGGCCGGTGTGGAGACAACGACTCCTTCCAAGCAGTCCAACAACAAATACGCAGCGAGTAGTTACCTGTCCCTAACCCCTGAGCAGTGGAAGTCGCACCGTTCATACTCGTGCCAGGTTACGCACGAGGGTTCAACGGTCGAAAAGACAGTAGCACCTACGGAATGCTCA44R24 HC coding sequence (SEQ ID NO: 91):ATGAAGCACCTGTGGTTCTTTCTGCTGCTGGTGGCCGCTCCTAGATGGGTGCTGTCCGAGGTGCAGCTGGTGGAGTCTGGCGGAGGACTGGTGCAGCCTGGCGGCTCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCTCTTACTACATCACCTGGGTGCGCCAGGCTCCTGGCAAGGGACTGGAATGGGTGTCCACCATCTCCTACTCCTCCAGCAACACCTACTACGCCGACTCCGTGAAGGGCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCCGGTCCATCGTGTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGTCCTCTTCCGTGTTCCCTCTGGCCCCTTGCTCCCGGTCCACCTCTGAGTCTACCGCCGCTCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTGACCGTGTCCTGGAACTCTGGCGCCCTGACCTCTGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGCCTTCCTCCAACTTCGGCACCCAGACCTACACCTGCAACGTGGACCACAAGCCTTCCAACACCAAGGTGGACAAGACCGTGGAGCGGAAGTGCTGCGTGGAGTGCCCTCCTTGTCCTGCTCCTCCTGTGGCTGGCCCTTCTGTGTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGGACCCCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAACAGTTCAACTCCACCTTCCGGGTGGTGTCTGTGCTGACCGTGGTGCACCAGGACTGGCTGAACGGCAAAGAATACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTGCCCCTATCGAAAAGACCATCTCTAAGACCAAGGGCCAGCCTCGCGAGCCTCAGGTCTACACCCTGCCTCCTAGCCGGGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAGTGGGAGTCTAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTATGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCCTGGCAAGTGA44R24 LC coding sequence (SEQ ID NO: 93):ATGGCTTGGGCTCTGCTGCTGCTGACCCTGCTGACACAGGGCACCGGCTCTTGGGCCGACATCGAGCTGACCCAGCCTCCCTCTGTGTCTGTGGCCCCTGGCCAGACCGCCAGGATCTCTTGCTCTGGCGACGCCCTGGGCAACAGATACGTGTACTGGTATCAGCAGAAGCCAGGCCAGGCCCCTGTGCTGGTGATCCCTTCCGGCATCCCTGAGCGGTTCTCCGGCTCCAACTCCGGCAACACCGCCACCCTGACCATCTCTGGCACCCAGGCCGAGGACGAGGCCGACTACTACTGCGGCTCCTGGGACACCCGGCCTTACCCTAAGTACGTGTTCGGCGGAGGCACCAAGCTGACCGTGCTGGGCCCTTCCGTGACCCTGTTCCCTCCATCCTCCGAGGAACTGCAGGCCAACAAGGCCACCCTGGTGTGCCTGATCTCCGACTTCTACCCTGGCGCCGTGACCGTGGCTTGGAAGGCCGACTCTAGCCCTGTGAAGGCCGGCGTGGAGACAACCACCCCTTCCAAGCAGTCCAACAACAAGTACGCCGCCTCCTCCTACCTGTCCCTGACCCCTGAGCAGTGGAAGTCCCACCGGTCCTACTCTTGCCAGGTGACCCACGAGGGCTCCACCGTGGAAAAGACCGTGGCCCCTACCGAGTGCTCCTAG

Plasmids isolated from E. coli encoding the anti-FZD IgG antibodies18R4605 (ATCC deposit no. PTA-10307), 18R4805 (ATCC deposit no.PTA-10309), and 44R24 (ATCC deposit no. PTA-10311) were deposited withthe American Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va., USA, under the conditions of the Budapest Treaty on Aug.26, 2009.

Example 19 Binding Profiles of Anti-FZD Antibodies

FACS analysis was used to characterize the FZD1, 2, 5, 7 and 8 bindingprofiles of anti-FZD monoclonal antibodies (mAbs).

HEK293 cells were co-transfected with a plasmid DNA expressingfull-length FZD1, 2, 5, 7 or 8 along with another plasmid expressing thereporter gene GFP used as a transfection marker. Fugene 6 (Roche) wasused as transfection reagent according to the manufacturer directions.Transfected cells were incubated twenty-four to forty-eight hours at 37°C. and 5% CO₂. The anti-FZD mAbs were then diluted in a final volume of50 μl starting with a concentration of 20 μg/ml and serially diluted4-fold for a total of 8 dilutions. Each FZD/GFP 293 transientlytranfectant pool was collected in suspension and 100,000 transfectedcells were incubated on ice 30-60 minutes with the diluted anti-FZD mAbto be tested. The cells were washed and bound anti-Fzd antibodies weredetected with a secondary anti-human antibody conjugated to afluorescent chromophore. Labeled cells were then detected and counted byFACS. The FACS data generated were expressed in Mean FluorescenceIntensity (MFI) units. GraphPad Prism software was used to graph andanalyze the data. MFIs were plotted as a function of Ab concentration toestablish dose-response curves. A non-linear regression was applied tothe numbers to fit the curve and calculate EC50s.

The binding profiles for the mAbs 18R5 and 44R24 were determined andcompared. The dose-reponse curve representing the binding of each of18R5 and 44R24 to Fzd1, 2, 5, 7 and 8 is shown in FIG. 47. The EC50s(nM) calculated for the two mAbs is shown in Table 13. 44R24 bound Fzd5and Fzd8 with good affinity. A sigmoidal dose-response curve could notbe established for the other 3 Fzd receptors, suggesting that 44R24 doesnot bind Fzd1, 2 and 7. High affinity binding of Fzd1, 2, 5 and 7 wasconfirmed for 18R5.

TABLE 13 EC50s (nM) for the mAbs 18R5 and 44R24 EC50 (nM) Fzd1 Fzd2 Fzd5Fzd7 Fzd8 18R5 0.41 0.62 1.10 0.58 12.00 44R24 117.95 no binding 1.8992.31 1.09

Example 20 Evaluation of Anti-Wnt Activity of Anti-FZD mAbs inCell-Based Assays

The ability of 18R5 and 44R24 to inhibit Wnt signaling in STF-293 cellswas determined and compared. STF cells are Human Embryonic Kidney(HEK)-293 cells stably transfected with the Super Top Flash (STF)reporter cassette in which the expression of the Luciferase (Luc)reporter gene is regulated by multiple copies of the TCF binding siteupstream of a minimal promoter. A low basal Luc expression can beinduced 30-60 fold in response to Wnt3a, providing a large window toassess the inhibitory activity of the anti-Fzd Abs.

To assess the mAbs, STF-293 cells were grown in DMEM-10% FBS. On day 1,10,000 cells were plated per well in 96-well Optical Bottom White plates(Nunc #165306). The cells were incubated O.N. at 37° C. and 5% CO₂. Onday 2, the Abs to be tested were diluted to a final concentration of 40μg/μl using culture medium. Seven 5-fold serial dilutions wereperformed. The STF-293 cells culture medium was replaced with a mixturecontaining 50 μl Ab dilution, 25 μl Wnt3a-conditioned medium from Wnt3astable L-cells and 25 μl DMEM-10% FBS. For each Ab, the finalconcentrations tested were 20, 4, 0.8, 0.16, 0.03, 0.006, 0.0013, 0.0003μg/ml. Each Ab concentration was tested in triplicate. A humananti-hapten Ab. LZ1, was used as negative control Ab. Anon-Wnt3a-conditioned medium from parental L-cells was used as negativecontrol inducer. The plates were returned to the incubator. Luciferaseactivity was measured on day 3, using Promega Steady Glo kit(VWR#PAE2550-A) according to the manufacturer specifications. Theresults were expressed in photons per sec. GraphPad Prism software wasused to graph and analyze the data. Luciferase activities were plottedas a function of Ab concentration to establish dose-response curves. Anon-linear regression was applied to the numbers to fit the curve andcalculate IC50s.

The ability of 44R24 and 18R5 to inhibit Wnt signaling in STF cells waslikewise determined and compared as described above. The results areshown in FIG. 48 and Table 14. 44R24 activity was only detected athigher Ab concentrations, reflecting the antibody's low activity in theassay. 44R24's IC50 was calculated to be 13 fold lower than 18R5's.

TABLE 14 IC50s for inhibition of Wnt signaling in ST-293 cells by 18R5and 44R24 18R5 44R24 IC50 (nM) 2.73 34.43

The ability of 18R5 and 44R24 to inhibit Wnt signaling in A549 cells wasalso determined. A549 cells are human lung carcinoma cells in which theAxin2 gene is highly expressed, translating endogenous activity of Wntsignaling. Axin2 is a well know Wnt target gene that responds toactivation of the pathway by up-regulating its transcription andeventually down-regulating Wnt signaling through a feed-back loopmechanism. This system was used to test the impact of the anti-Fzd Abson Axin2 mRNA levels by qPCR.

12-well plates were seeded with 30,000 A549 cells per well and grown for3 days in DMEM+10%FBS. Antibodies were added at varying concentrations(5 μl, 0.2, 0.04, 0.008 μg/ml) for 24 hours and total RNA was extractedfrom the cells. LZ1, a nonbinding antibody, was used as negative controlat only the highest concentration.

30,000 A549 cells were seeded into 12-well plates and grown for 3 daysin DMEM+10% FBS. Anti-FZD antibody 18R5 or 44R24 were added at varyingconcentrations (5, 1, 0.2, 0.04, 0.008 ug/ml), and LZ1, a nonbindingantibody, was used as a negative control at only the highestconcentration. RNA was made 24 hours post treatment and then treatedwith Dnase.

Axin2 is known to be a robust target gene in Wnt signaling and itsexpression level was examined by doing a Taqman relative expression(ΔΔCT) assay using an Applied Biosystems 7900 HT machine. 50 ng of RNAwas used per point in triplicate and a GUSB probe was used forendogenous control. All results were normalized to Axin2 levels in theLZ1 control sample.

The dose response curve showing the inhibition of basal level of axin2gene expression by 18R5 and 44R24 and the calculated EC50 values forthese antibodies are shown in FIG. 49. 18R5 and 44R24 inhibit Axin2basal levels relative to LZ1 control with comparable efficiencies.

Example 21 Evaluation of Anti-Tumor Activity of Anti-FZD mAbs inPancreatic Xenograft Models

OMP-PN13 pancreatic tumors:

Frozen OMP-PN13 tumor cells that have been passaged twice in mice wereobtained from Oncomed's tumor bank. They were thawed and injectedsubcutaneously into the left flank of NOD/SCID mice immediatelyfollowing thawing. ˜25,000 viable cells were injected per animal. Themice were monitored weekly for tumor growth. After their onset, the sizeof the tumors was measured once weekly by caliper. 200-300 mm³tumor-bearing mice were dispatched in treatment groups that eachcontained 5 animals. The average tumor size was comparable in eachgroup. Ab treatment was initiated the day after randomization. LZ1 wasused as the negative control Ab. 3 doses of 10 mg/kg of Ab wereadministered via intra-peritoneal injection over a 12-day period. Micewere euthanized 24 hours after the last injection. Tumor, duodenum andliver were harvested.

Tumor tissues were fixed in formalin for paraffin embedding andsectioning. Muc16 detection was performed by immunohistochemical (IHC)to monitor the appearance of Mucin-producing cells. Mucins are specificfor a subtype of differentiated cells in the pancreas, and as such areused as differentiation markers in the tumor model.

Formalin-fixed, paraffin-embedded (FFPE) sections were de-parrafinized.The slides were first de-parrafinized by sequential treatment withxylene twice for 5 minutes each. The tissue was then rehydrated byimmersion in an ethanol series of 100% twice for 3 minutes each, 90%once for 1 minute, 80% once for 1 minute, and 70% once 1 minute inwater. The tissue was washed with flowing distilled water for 1 minute.

Mucin 16 antibody (clone X325 from AbCAM, catalog ab10033) was used forIHC detection of mucin 16 expressing cells in FFPE tissue sections.Heat-induced antigen retrieval was carried out with 10 mM citrate bufferpH 6.0 in an autoclave. The slides were then kept at room temperatureand the proteins allowed to recover antigenicity slowly (approximately 2hours).

Tissue sections were blocked with 3% hydrogen peroxide solution inwater, washed, and then blocked again using Normal Horse serum Blockingsolution (for 50 mL; PBS (38.5 mL),10% NHS (5 mL),1% BSA (5 mL), 0.1%gelatin (500 μL), 0.1% Tx-100 (500 μL), 0.05% NaN3 (500 μL)) for 1 hourat room temperature. Sections were then stained with 1:200 dilution ofMuc16 primary antibody in Da Vinci Green Diluent pH 7.3 (PD 900, BiocareMedical) for 1 hour at room temperature followed by three washes usingphosphate buffered saline containing 0.1% triton X-100. Sections werethen stained with 3 drops of ImmPress anti-mouse IgG HRP-conjugate(Catalog 101098-260, VWR) for 30 minutes at room temperature followed bythree washes using phosphate buffered saline containing 0.1% tritonX-100. Slides were placed in petridishes and developed using VectorNovaRed kit (SK4800, Vector labs) for 1-2 minutes. Reaction was stoppedby adding distilled water. Slides were rinsed thoroughly under flowingdistilled water. Tissue sections were then counterstained usingHematoxylin (Catalog H3401, Vector Labs Gill's formula) for 1 minute,washed, and then neutralized using blueing solution for 30 seconds.Slides were left to dry overnight and mounted using VectaMount (VectorLabs).

Representative fields of tumors treated with control Ab (LZ1), 18R5, or44R24 are shown in FIG. 50. While LZ1 was associated with low intensitystaining, higher levels of staining with Muc16 antibody were detected inthe tumors treated with 18R5. This suggests that tumor cells wereinduced to differentiate towards the mucin-producing cell lineage by18R5. Levels of Muc16 staining in the 44R24-treated tumors were moremoderate than in the 18R5 treated tumors but still appeared to beslightly higher than in the LZ1-treated tumors in this experiment.

Total RNAs were also extracted from tumor, duodenum and liver for Wnttarget gene expression analyses using qPCR.

Tissues were immediately transferred into RNAlater (QIAGEN) at the timeof harvest. RNA was extracted using the QIAGEN RNeasy for Fibrous Tissuemini kit according to the manufacturer instructions. 50 ng total RNAwere submitted to gene expression analyses using ABI one-step RT-PCRprotocol and reagents. GusB gene expression was used as endogenouscontrol. Triplicates were setup for each sample. All 5 tumors of eachtreatment group were analyzed. ABI 7900 TaqMan machine was used to runthe experiments. ABI SDS 2.2.1 software was used to analyze the data andcalculate DeltaCt values that were converted into relative quantities.The triplicate values of all 5 tumors were averaged for each treatmentgroup. Fold inhibition factors were then calculated relative to controlantibody (LZ1).

The results are shown in Table 15. Wnt target genes were variablyaffected by the anti-FZD Abs. 18R5 induced 2.3× and 8× inhibition intumor and liver, while remaining unaffected in the duodenum. The changesinduced by 44R24 were more moderate.

TABLE 15 qPCR gene expression analysis of Wnt target genes in 18R5- and44R24-treated tissues. tumor Liver duodenum Axin2 Axin2 Rhbg GluI Lect2Lgr5 Axin2 Lgr5 18R5 −2.3x  −8x −6.5x −19x −8x −26x NS −2.6x −2.5x −13x−5x 44R24 −1.8x  −2.4x −1.9x ND ND ND ND ND ND: not done Separateexperiments are shown in italics

OMP-PN4 Pancreatic Tumors:

The impact of 18R5 on tumor stroma in the OMP-PN4 pancreatic tumorxenograft model was also investigated. Several genes were identified bymicroarray whose expression levels were altered by the treatment.Amongst them, ACTA2, which encodes for the smooth muscle actin (SMA)protein, is of particular interest. SMA has been shown to be associatedwith activated tumor stroma. Its down-regulation can therefore be viewedas a sign of decreased tumorgenic phenotype.

As described in Example 7 above, OMP-PN4 tumor-bearing NOD/SCID micewere treated with control Ab (LZ-1). 18R5, gemcitabine, or thecombination of 18R5 and gemcitabine once a week for 6 weeks. Theantibodies were administered at a concentration of 10 mg/kg. After theywere harvested, the tumors from that experiment were analyzed for theexpression of Wnt target genes at both the RNA and protein levels, usingmicroarray and IHC, respectively.

Total RNA was extracted from the tumors, amplified, and subjected tomicroarray analysis. Total RNAs were amplified using the Ovation RNAAmplification System V2 (NuGEN, San Carlos, Calif.). Resultingamplified, antisense ss-cDNA was fragmented and biotinylated using theFL-Ovation cDNA Biotin Module V2 (NuGEN) for use on Affymetrix chips.Affymetrix HG-U133 plus 2 or MG 430 2.0 oligonucleotide microarrays wereused in these experiments (performed at Almac Diagnostics, Durham,N.C.). After hybridization, Gene Chips were washed, stained, and scannedaccording to the manufacturer's instructions (Affymetrix, Santa Clara,Calif.). The quality of the cDNA and the fragmented cDNA were assessedby spectrophotometer and the Bioanalyzer before the array hybridization.The scanned raw chip data were quantified and scaled using the GCOSsoftware package (Affymetrix) and subjected to a comprehensiveassessment of Gene Chip Quality Control recommended by Affymetrix todetect any chips defects and outliers, which were excluded from thesubsequent data analysis.

Array background adjustment and signal intensity normalization wereperformed with GCRMA algorithm in the open-source Bioconductor software(www.bioconductor.org). Genes differentially expressed between twogroups or time points were identified with Bayesian t-test (Cyber-T),which combines student's t-test with a Bayesian estimate of theintra-group variance obtained from the observed variance of probe setsat a similar expression levels (Baldi P, Long AD. A Bayesian frameworkfor the analysis of microarray expression data: regularized t-test andstatistical inferences of gene changes. Bioinformatics. 2001;17(6):509-19).

For the human tumor gene chip analysis, samples were assayed on bothhuman and mouse chips to assess treatment effects on bulk human tumorand on mouse stroma independently. Those Affymetrix probe sets that werenot species-specific were omitted from the analysis.

In preparation for Smooth Muscle Actin alpha (SMAa) immunofluorescence,tumor tissue was frozen using OCT. 4 micron sections were obtained andstored frozen at −80° C. For SMAa staining, tissue was fixed usingchilled acetone at −20° C. for 15 minutes and then allowed to dry andcome to room temperature and then marked using a hydrophobic PAP pen.Slides were then washed using phosphate buffered saline (PBS). Tissuewas blocked using normal horse serum R.T.U. (Vector Labs) for 2 hours atroom temperature. Primary antibody staining was performed with 1:10,000dilution of FITC-conjugated Smooth muscle actin alpha antibody (cline1A4, #F3777, SIGMA) for 1 hour. Sections were washed 3 times using PBScontaining 0.1% triton X-100. Slides were then air-dried and thenmounted using Hard set mounting medium containing DAPI (vectashiedH-500).

FIG. 51A shows the ACTA2 gene expression levels as detected bymicroarray. Tumors treated with the anti-FZD antibody 18R5 showeddecreased levels of ACTA2 expression. FIG. 51B shows the results of theSmooth Muscle Actin alpha (SMAa) immunofluorescence on control mAb(upper panel) and 18R5 (lower panel)-treated OMP-PN4 tumors. Reducedamounts of SMAa was detected on the 18R5-treated tumors. The expressionof ACTA2 and the amount of SMA were dramatically reduced in the tumorstroma in response to 18R5, suggesting that Wnt blockade (i) impactsthis tumor compartment and (ii) does so by reducing a well establishedtumorgenicity marker. These results suggest that reduction ofmyofibroblast activation may be one of 18R5's anti-tumor mechanisms ofaction.

Example 22 Evaluation of the Tumorgenic Potential of Muc 16-PositveOMP-PN 13 Cells

As described above, 18R5 treatment induced gene expression and cellphenotype changes in pancreatic tumors, including increased mucinexpression. In particular, IHC performed on treated PN-13 tumorsrevealed an increased number of Muc16-positive cells. Muc16 geneexpression levels were also higher in treated tumors than in controltumors. The tumorigenicity of the 18R5-induced Muc16-positive cells wasassessed to test the hypothesis that the 18R5-induced Muc16-positivecells are representative of a differentiated tumor cell sub-population.

OMP-PN13-bearing mice were treated with 18R5 according to the protocoldescribed in Example 21. Mice were euthanized 12 days after initiationof the Ab treatment. Tumors were harvested and processed to obtain asingle cell suspension using collagenase III to digest the tissues.Mouse stromal cells were stained with a biotinylated anti-H-2 Kdantibody and a biotinylated anti-CD45 antibody. They were then incubatedwith magnetic beads conjugated to streptavidin (Thermo MagnaBind) anddepleted using a Dynal magnet. The resulting lin-depleted tumor cellswere stained with an anti-Muc16 mAb detected with a PE-conjugatedsecondary Ab. Muc16-positive and Muc16-negative cells were sorted usingan ARIA FACS machine ran by the DIVA software. See FIG. 52A. Cells werere-injected subcutaneously in the left flank of NOD/SCID mice forcomparison of their tumorgenic potentials. Each cell type was injectedinto 10 mice. Each mouse received 75 cells. Representative pictures oftumors resulting from the injection of Muc16-(upper panel) and Muc16+(lower panel) cells are shown in FIG. 52B. The growth curves for theMuc16− and Muc16+ tumors following injection into the mice are shown inFIG. 52C. No tumor grew after injection of the Muc16+ cells, while 7 ofthe 10 mice injected with the Muc16-cells developed tumors. The datasuggest that 18R5-induced Muc16+ cells are non-tumorgenic, supportinginduction of differentiation as an underlying mechanism of 18R5anti-tumor activity.

Example 23 Additional In Vivo Studies with Anti-FZD Antibodies

PE13 breast tumor recurrence study with 18R5 mAb: PE13 breast tumorcells were injected into Nod-Scid mice and allowed to grow until thetumors had reached approximately 100 mm³. The animals were randomizedinto two groups (n=10) and given taxol (15 mg/kg, twice perweek)+Control antibody (black squares) or the same dose oftaxol+Anti-FZD 18R5 (gray open circles). Antibodies were dosed at 20mg/kg once per week. Taxol® treatments were stopped at day 70 and theantibody treatments continued. The results are shown in FIG. 53. 18R5was observed to enhance the rate of tumor regression and to delay tumorrecurrence after stopping taxol treatment.

PE13 breast tumor limiting dilution assay (LDA) study with 18R5 mAb:Animals bearing PE13 breast tumors were treated with either controlantibody (gray circles). 18R5 (open triangles), taxol (black circles),or the combination of taxol and 18R5 (open squares). Taxol® was dosed at15 mg/kg twice per week and the antibodies were dosed 20 mg/kg once perweek. Tumors were harvested and the human tumor cells were purified bylin depletion. 50, 150, or 500 tumor cells were injected into a newcohort of mice (n=10 per cell dose). The results are shown in FIG. 54.Tumor growth frequency was monitored after 59 days and used to calculatethe CSC frequency (L-calc).

PN4 pancreatic tumor recurrence study with 18R5 mAb: PN4 pancreatictumor cells were injected into Nod-Scid mice and allowed to grow untilthe tumors had reached approximately 250 mm³. The animals were givengemcitabine (75 mg/kg, once per week) for 5 weeks until tumors hadregressed. The animals were randomized into two groups and given controlantibody (black squares) or anti-FZD 18R5 (gray open circles).Antibodies were dosed at 10 mg/kg once per week. The results are shownin FIG. 55. 18R5 was observed to delay tumor recurrence aftergemcitabine treatment.

PN4 pancreatic tumor growth study with 44R24 mAb: PN4 pancreatic tumorswere injected into Nod-Scid mice. Tumors were allowed to grow until theyhad reached a volume of approximately 150 mm³. Animals were randomizedinto 4 groups (n=10 per group) and given control antibody (blacksquares), anti-FZD5/8 44R24 (gray open triangles), gemcitabine (filledtriangles), or combination of 44R24 plus gemcitabine (gray opencircles). Gemcitabine was dosed at 15 mg/kg once per week and theantibodies were dosed at 20 mg/kg twice week. The results are shown inFIG. 56. 44R24 was observed to reduce tumor growth in combination withgemcitabine relative to gemcitabine alone.

Example 24 Epitope Mapping of Anti-FZD Antibody 44R24

Epitope mapping for the anti-FZD antibody 44R24 was performed in amanner similar to that described above in Example 5 for the antibodies18R8 and 18R5. The ability of 44R24 to bind to a similar epitope as 18R8was assessed by flow cytometry using a series of amino acid variants ofFZD8 previously shown to disrupt binding of 18R8 (see Example 5 andFIGS. 6 and 7). Amino acids 126-127 of FZD8 were found to be requiredfor binding 44R24 as indicated by reduced staining within theco-transfected (GFP positive) cell population. The results of the FACSexperiments are shown in FIG. 57. These results show that 44R24 binds toan epitope that overlaps with the epitope of 18R8 and comprises sharedamino acids 126-127.

Example 25 C28 Colon Tumor Growth Study with Anti-FZD Antibodies 18R5,18R8 and 44R24

C28 tumor cells were injected sub-cutaneously in Nod-Scid mice. Tumorswere allowed to grow until they had reached an average volume of 126mm³. Tumor bearing animals were groups were randomized into four groups(n=10 mice per group) and treated with either Control Ab (blacksquares), 18R8 (gray triangles), 44R24 (black open circles), or 18R5(gray circles) (FIG. 58). Antibodies were dosed intraperitoneally at 15mg/kg, twice per week. Tumor volumes are indicated. Treatment withantibodies 44R24 and 18R5 reduced growth relative to the control group,while 18R8 had no effect (FIG. 58).

Following treatment of the animals in the experiment shown in FIG. 58,tumors were harvested, fixed in formalin, embedded in paraffin, and cutin 5 micron sections. Tumor sections were analyzed for Cytokeratin 7expression, a marker of colon cell differentiation, byimmunohistochemistry (Vectastain kit, Vector Labs). Cytokeratin 7expression was seen to be elevated after treatment with 44R24 or 18R5(FIG. 59).

Example 26 Production and Purification of 18R5

A CHO-derived recombinant cell line expressing the 18R5 antibody wasproduced using standard methods known to one of skill in the art. Forantibody production and purification, the cell line was cultured using afed-batch bioreactor cell culture process and was grown inchemically-defined and serum-free media. The cell culture process wasrun for about 10-15 days and included a temperature shift from 37° C. to34° C. at approximately 5 days post-inoculation.

The 18R5 antibody was purified in a multistep purification scheme.First, harvested cell culture fluid (HCCF) from the bioreactor(s) waspurified by affinity chromatography using MabSelect SuRe™ (GE HealthcareLife Sciences). The HCCF was applied to the MabSelect SuRe™ column at25-30 grams of protein per liter of resin and the column wassubsequently washed three times. Bound 18R5 antibody was eluted from thecolumn with 100 mM glycine-HCl (pH 3.2). The 18R5-containing eluate washeld at low pH for 1 hour at room temperature for viral inactivation,followed by an adjustment of the pH to about 6.5 using 1M Bis-Trisbuffer (pH 8.5). The eluate was then filtered through a 0.2 micronfilter and stored at 2-8° C. until the next purification step.

Second, the 18R5-containing eluate was subjected to anion exchangechromatography using Capto Q resin (GE Healthcare Life Sciences). The18R5 antibody flowed through the Capto Q column and was collected, whileimpurities bound to the column and were removed from the 18R5 pool. Theflow-through pool containing the 18R5 antibody was filtered through a0.2 micron filter and stored until the next purification step.

Third, the 18R5-containing flow-through pool was subjected to ceramichydroxyapatite (CHT) chromatography on a packed CHT type I column(BioRad Laboratories). The 18R5 antibody and remaining impurities werebound to the CHT column. The column was washed two times with low saltbuffers (0-100 mM NaCl) containing 5-10% polyethylene glycol (PEG). The18R5 antibody was eluted from the column with a high salt buffer(400-1000 mM NaCl) containing 5-10% PEG and collected. Tangential flowultrafiltration and diafiltration was used to exchange the high saltelution buffer with a buffer comprising histidine, NaCl and sucrose. Thepurified 18R5 antibody was filtered through a 0.2 micron filter andstored at 2-8° C. for future use.

Example 27 Residues within the OMP-18R5 Binding Site Impact Wnt Binding

As noted above, the 18R5 antibody binds to the Frizzled receptors byinteraction via a discontinuous epitope. The surface of the FZD receptorwithin the 18R5 binding area contains a cleft, apparent in the crystalstructure of FZD (FIG. 9 and FIG. 60A). The amino acid residues withinthe cleft of the BBS of each of the human FZD receptors are provided inTable 16. The residues that line the base of this cleft exhibitparticularly high conservation between FZD family members suggestingfunctional importance. 18R5 binding to FZD blocks the interaction of Wntto FZD demonstrating that the region is functionally relevant for Wntsignaling. To further explore the importance of residues within thisbinding region in FZD8, select residues that line the cleft were mutatedto alternative residues (FIG. 60B). (In certain alternative embodiments,the analogous residues within another human FZD, as identified in Table17 below, may instead be used in a screening method, as may otherresidues within the BBS). The ability of FZD receptors containing thesesubstitutions to bind Wnt3A was then assessed (FIG. 60C). Each of thesubstitutions greatly decreased the ability of Wnt to bind to the FZDreceptors confirming the importance of this region of the FZD receptorin mediating Wnt interaction.

TABLE 16 Amino acid residues in “cleft” of the Biological Binding Sites(BBS) of FZD Receptors Human Frizzled Receptor (aa sequence) BBS CleftResidues FZD1 153(F), 155-156(PL), 159(V), 173(Y), (SEQ ID NO: 26)201-202(LM), 209-212(WPDT) FZD2 76(F), 78-79(PL), 82(V), 96(Y), (SEQ IDNO: 30) 124-125(LM), 132-135 (WPER) FZD3 65(F), 67-68(PM), 71(L), 85(Y),(SEQ ID NO: 34) 113-114(LM), 121-124 (WPED) FZD4 85(F), 87-88(PL),91(Y), 105(Y), (SEQ ID NO: 38) 134-135(VL), 142-145(WPES) FZD5 70(F),72-73(PL), 76(I) 90(Y), (SEQ ID NO: 42) 119-120(LM), 127-130(WPER) FZD661(F), 63-64(PL), 67(L), 81(F), (SEQ ID NO: 46) 109-110(LI),117-120(WPEE) FZD7 86(F), 88-89(PL), 92(V), 106(Y), (SEQ ID NO: 50)134-135(LM), 142-145(WPER) FZD8 72(F), 74-75(PL), 78(I), 92(Y), (SEQ IDNO: 54) 121-122(LM), and 129-132(WPDR) FZD9 76(F), 78-79(PL), 82(Y),96(Y), (SEQ ID NO: 58) 125-126(IM), 133-136(WPDS) FZD10 71(F),73-74(PL), 77(Y), 91(Y), (SEQ ID NO: 62) 120-121(IM), 128-131(WPDS)

TABLE 17 Amino acid position of analogous residues of mutants in the tenhuman FZD receptors Frizzled Mut1 Mut2 Mut3 Mut4 FZD1 153 156 159 201FZD2 76 79 82 124 FZD3 65 68 71 113 FZD4 85 88 91 134 FZD5 70 73 76 119FZD6 61 64 67 109 FZD7 86 89 92 134 FZD8 72 75 78 121 FZD9 76 79 82 125FZD10 71 74 77 120

Example 28 Identification of Small Molecule Peptides that Bind the WntBinding Region of FZD

To identify small molecule peptides that bind to FZD we screened peptidelibraries (Ph.D. phage display peptide library system, New EnglandBiolabs). The libraries were screened using the URSA method (Ultra RapidSelection of Antibodies) using recombinant FZD8-Fc bound to protein Abeads. After three rounds of enrichment, individual clones were testedfor binding to FZD. As shown in FIG. 61, peptides were identified thatbound to FZD but not to an irrelevant control protein (DLL4-Fc).demonstrating that they specifically interact with FZD. Representativepositive clones are highlighted by arrows. The sequences of the FZDbinding peptides were determined. Both linear peptides from a library of12-mer peptides and cyclic peptides from a library comprised of sevenrandom amino acids flanked by cysteine residues were isolated. As isshown in FIG. 62, the cyclic peptides displayed similar but distinctsequences suggesting that they adopt similar structure and demonstrateda clear consensus sequence.

To examine whether any of these FZD binding peptides interacted with FZDthrough the BBS, we examined the ability of the peptides to interactwith FZD with amino acid substitutions that were shown in theexperiments described above in Example 27 (FIG. 60C) to impact Wntbinding. As is shown in FIG. 63, FZD binding peptides were identifiedthat specifically interact with FZD within the Wnt binding region.Several peptides displayed greatly reduced or eliminated binding to theFZD when an amino acid substitution was introduced within the cleftregion, demonstrating the ability to identify small molecules thatinteract with this region.

Example 29 Inhibition of Wnt Signaling by Small Molecules that Bind theWnt Binding Region of Frizzled

To examine whether the peptides that bind the FZD extracellular domainand interacted with the BBS region were able to impact Wnt signaling,Wnt reporter assays were conducted. Two peptides were chemicallysynthesized containing the sequence of clones #2 and #24. The Wntreporter assay utilized an 8xTCF luciferase reporter that is inducibleby exogenous Wnt proteins that activate canonical Wnt signaling. Asshown in FIG. 64, small peptides that bind to FZD protein were able toinhibit the ability of HEK293 cells to respond to Wnt3A.

Example 30 Identification of Non-Peptide Small Molecules that Bind tothe FZD BBS Wnt Binding Region

There are many ways in which one can identify small molecules that bindto the Wnt binding region of the FZD receptors. In one approach, one canmake use of peptides that bind within the BBS, or more specifically, thecleft region of FZD to enable rapid and high throughput screens of smallmolecule chemical libraries. As one non-limiting example, the peptidecan be labeled with a convenient reporter (e.g. radiolabel, fluorescenttag, or FRET tag). The labeled peptide can then be allowed to bind toFZD protein. The peptide-FZD complex can then be allowed to interactwith a test compound from a small molecule library and the ability ofthe small molecule to displace the peptide from the FZD receptor orcompete with the peptide for binding to the FZD protein can be measured.In such a manner one is able to identify small molecules that bindspecifically to the FZD protein within the desired binding region.

Example 31 Treatment of Polycystic Kidney Disease with 18R5

The ability of 18R5 to treat polycystic kidney disease (PKD) can bedemonstrated in murine models of PKD. There are several murine models ofPKD which reflect the underlying mutations observed in human PKD anddemonstrate pathology very comparable to the human disease (referencesbelow). Several of these models involve alteration in the levels ofexpression of either the PKD1 gene which encodes polycystin1 (PC1) orthe PKD2 gene which encodes polycystin2 (PC2). These genes are mutatedin almost all human cases of polycystic kidney disease. Utilizing onesuch model, one treats mice that possess altered expression of PKD1 orPKD2 (and are subsequently prone to develop PKD) by administration of18R5 via IP injection at a dose of 15 mg/kg twice per week. After onemonth of treatment one can observe improvement in kidney functionrelative to control mice not treated with 18R5. This improved functionis manifested by decreased size and abundance of kidney cysts, reducedproliferation of kidney epithelial cells as measurable by ki-67 IHCstaining of tissue sections, and improved kidney function as measured byreduced blood urea concentrations. Relevant references include Happé etal. Hum Mol. Genet. 2009 Jul. 15; 18(14):2532-42, Kim et al., J Am SocNephrol. 2009 December; 20(12):2556-69, and Kurbegovic et al., Hum Mol.Genet. 2010 Apr. 1; 19(7):1174-89, each of which is hereby incorporatedby reference herein in its entirety.

Example 32 Additional Purification Scheme for Monoclonal Antibody 18R5

A CHO-derived recombinant cell line expressing the 18R5 antibody wasproduced using standard methods known to one of skill in the art. Forantibody production and purification, the cell line was cultured using afed-batch bioreactor cell culture process and was grown inchemically-defined and serum-free media. The cell culture process wasrun for about 10-15 days and included a temperature shift from 37° C. to34° C. at approximately 5 days post-inoculation.

The 18R5 antibody was purified in a multistep purification scheme.First, harvested cell culture fluid (HCCF) from the bioreactor(s) waspurified by affinity chromatography using MabSelect SuRe™ (GE HealthcareLife Sciences). The HCCF was applied to the MabSelect SuRe column at25-30 grams of protein per liter of resin and the column wassubsequently washed three times. Bound 18R5 antibody was eluted from thecolumn with 100 mM glycine-HCl (pH 3.2). The 18R5-containing eluate washeld at low pH for 1 hour at room temperature for viral inactivation,followed by an adjustment of the pH to about 6.75-6.85 using 0.5-1MBis-Tris buffer (pH 8.5). The eluate was then filtered through a 0.2micron filter and stored at 2-8° C. until the next purification step.

Second, the 18R5-containing eluate was subjected to anion exchangechromatography using Capto Q resin (GE Healthcare Life Sciences). The18R5 antibody flowed through the Capto Q column and was collected, whileimpurities, such as aggregate, bound to the column and were removed fromthe 18R5 pool. The flow-through pool containing the 18R5 antibody wasfiltered through a 0.2 micron filter and stored until the nextpurification step.

Third, the 18R5-containing flow-through pool was subjected to ceramichydroxyapatite (CHT) chromatography on a packed CHT type I column(BioRad Laboratories). The 18R5 antibody and remaining impurities, suchas aggregates, were bound to the CHT column. The column was washed twotimes with low salt buffers (0-100 mM NaCl) containing 0-10%polyethylene glycol (PEG). The 18R5 antibody was eluted from the columnwith a high salt buffer (400-1000 mM NaCl. such as, for example, 546 mM)containing 10-13% PEG (such as, for example, 12.5% PEG) and collected.Tangential flow ultrafiltration and diafiltration were used to exchangethe high salt elution buffer with a buffer comprising histidine andNaCl. After diafiltration, the 18R5-containing solution was diluted withan excipient buffer comprising histidine, NaCl, sucrose andpolysorbate-20. The purified 18R5 antibody was filtered through a 0.2micron filter and stored at 2-8° C., or frozen at −65° C. or below, forfuture use.

All publications, patents, patent applications, internet sites, andaccession numbers/database sequences (including both polynucleotide andpolypeptide sequences) cited herein are hereby incorporated by referencein their entirety for all purposes to the same extent as if eachindividual publication, patent, patent application, internet site, oraccession number/database sequence were specifically and individuallyindicated to be so incorporated by reference In addition, U.S. patentapplication Ser. No. 12/568,534, filed on Sep. 28, 2009, which publishedas U.S. Patent Publication No. 2010/0104574, is hereby incorporated byreference herein in its entirety.

1-60. (canceled)
 61. An isolated polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NOs:98-116. 62.The polypeptide of claim 61, a) which is less than 100 amino acids inlength; b) which is less than 20 amino acids in length; c) which has amolecular weight of less than 2000 Daltons; d) which is a modifiedpolypeptide; e) which is labeled; f) which is a cyclic polypeptide; g)which is a linear polypeptide; and/or h) which is an inhibitor of Wntsignaling.
 63. The polypeptide of claim 61, which consists of an aminoacid sequence selected from the group consisting of SEQ ID NO:98-116.64. The polypeptide of claim 61, which comprises the amino acid sequenceof SEQ ID NO:112.
 65. The polypeptide of claim 61, which comprises theamino acid sequence of SEQ ID NO:108.
 66. The polypeptide of claim 61,which binds to a human frizzled (FZD) receptor.
 67. The polypeptide ofclaim 66, wherein the human FZD receptor is FZD8.
 68. The polypeptide ofclaim 61, wherein a) the polypeptide inhibits the binding of a Wnt to ahuman FZD receptor, or b) the polypeptide inhibits the binding of Wnt3Ato the human FZD8 receptor.
 69. The polypeptide of claim 61, wherein thepolypeptide inhibits Wnt signaling.
 70. A pharmaceutical compositioncomprising the polypeptide of claim
 61. 71. An agent comprising thepolypeptide of claim
 61. 72. An agent that competes with the polypeptideof claim 61 for binding to a human FZD receptor.
 73. The agent of claim72, wherein the FZD receptor is FZD8.
 74. The agent of claim 72, a)which is an antibody b) which is a polypeptide that is not an antibody;c) which comprises a polypeptide that is less than 100 amino acids inlength; d) which comprises a polypeptide that is from 5 to 20 aminoacids in length; e) which comprises a polypeptide that is labeled,glycosylated, pegylated, phosphorylated, acetylated, or amidated; f)which comprises a lipid; or g) which is a small molecule.
 75. A methodof inhibiting Wnt signaling in a cell, comprising contacting the cellwith an effective amount of the polypeptide of claim
 61. 76. The methodof claim 75, wherein the cell is a tumor cell.
 77. A method of inducingdifferentiation of a cell, comprising contacting the cell with aneffective amount of the polypeptide of claim
 61. 78. The method of claim77, wherein the cell is a tumor cell.
 79. A method of inhibiting growthof a tumor, comprising contacting the tumor with an effective amount ofthe polypeptide of claim
 61. 80. A method of treating a diseaseassociated with Wnt signaling activation comprising administering to asubject an effective amount of the polypeptide of claim
 61. 81. Themethod of claim 80, wherein the disease is cancer.